Chimeric factor viii polypeptides and uses thereof

ABSTRACT

The present invention provides a VWF fragment comprising the D′ domain and D3 domain of VWF, a chimeric protein comprising the VWF fragment and a heterologous moiety, or a chimeric protein comprising the VWF fragment and a FVIII protein and methods of using the same. A polypeptide chain comprising a VWF fragment of the invention binds to or is associated with a polypeptide chain comprising a FVIII protein and the polypeptide chain comprising the VWF fragment can prevent or inhibit binding of endogenous VWF to the FVIII protein. By preventing or inhibiting binding of endogenous VWF to the FVIII, which is a half-life limiting factor for FVIII, the VWF fragment can induce extension of half-life of the FVIII protein. The invention also includes nucleotides, vectors, host cells, methods of using the VWF fragment, or the chimeric proteins.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/371,948, filed Jul. 11, 2014, which is a 35 U.S.C. § 371 filing ofInternational Patent Application No. PCT/US2013/021330, filed Jan. 12,2013, which claims priority to U.S. Provisional Patent Application Ser.Nos. 61/734,954, filed Dec. 7, 2012; 61/667,901, filed Jul. 3, 2012;61/586,654, filed Jan. 13, 2012; and 61/586,099, filed Jan. 12, 2012,the entire disclosures of which are hereby incorporated herein byreference.

BACKGROUND OF THE INVENTION Sequence Listing

The content of the electronically submitted Sequence Listing in ASCIItext file (Name: 722088_SA9-417USCON2_ST25.txt; Size: 288,457 bytes;Date of Creation: May 27, 2022) is incorporated herein by reference inits entirety.

Coagulation is a complex process by which blood forms clots. It is animportant part of hemostasis, the cessation of blood loss from a damagedvessel, wherein a damaged blood vessel wall is covered by a platelet andfibrin-containing clot to stop bleeding and begin repair of the damagedvessel. Disorders of coagulation can lead to an increased risk ofbleeding (hemorrhage) or obstructive clotting (thrombosis).

Coagulation begins almost instantly after an injury to the blood vesselhas damaged the endothelium lining of the vessel. Exposure of the bloodto proteins such as tissue factor initiates changes to blood plateletsand the plasma protein fibrinogen, a clotting factor. Plateletsimmediately form a plug at the site of injury; this is called primaryhemostasis. Secondary hemostasis occurs simultaneously: Proteins in theblood plasma, called coagulation factors or clotting factors, respond ina complex cascade to form fibrin strands, which strengthen the plateletplug. Non-limiting coagulation factors include, but are not limited to,factor I (fibrinogen), factor II (prothrombin), Tissue factor, factor V(proaccelerin, labile factor), factor VII (stable factor, proconvertin),factor VIII (Antihemophilic factor A), factor IX (Antihemophilic factorB or Christmas factor), factor X (Stuart-Prower factor), factor XI(plasma thromboplastin antecedent), factor XII (Hageman factor), factorXIII (fibrin-stabilizing factor), VWF, prekallikrein (Fletcher factor),high-molecular-weight kininogen (HMWK) (Fitzgerald factor), fibronectin,antithrombin III, heparin cofactor II, protein C, protein S, protein Z,plasminogen, alpha 2-antiplasmin, tissue plasminogen activator (tPA),urokinase, plasminogen activator inhibitor-1 (PAI1), and plasminogenactivator inhibitor-2 (PAI2).

Haemophilia A is a bleeding disorder caused by defects in the geneencoding coagulation factor VIII (FVIII) and affects 1-2 in 10,000 malebirths. Graw et al., Nat. Rev. Genet. 6(6): 488-501 (2005). Patientsaffected with hemophilia A can be treated with infusion of purified orrecombinantly produced FVIII. All commercially available FVIII products,however, are known to have a half-life of about 8-12 hours, requiringfrequent intravenous administration to the patients. See Weiner M. A.and Cairo, M. S., Pediatric Hematology Secrets, Lee, M. T., 12.Disorders of Coagulation, Elsevier Health Sciences, 2001; Lillicrap, D.Thromb. Res. 122 Suppl 4:S2-8 (2008). In addition, a number ofapproaches have been tried in order to extend the FVIII half-life. Forexample, the approaches in development to extend the half-life ofclotting factors include pegylation, glycopegylation, and conjugationwith albumin. See Dumont et al., Blood. 119(13): 3024-3030 (Publishedonline Jan. 13, 2012). Regardless of the protein engineering used,however, the long acting FVIII products currently under development haveimproved half-lives, but the half-lives are reported to be limited—onlyto about 1.5 to 2 fold improvement in preclinical animal models. See Id.Consistent results have been demonstrated in humans, for example,rFVIIIFc was reported to improve half-life up to ˜1.7 fold compared withADVATE® in hemophilia A patients. See Id. Therefore, the half-lifeincreases, despite minor improvements, may indicate the presence ofother T½ limiting factors. See Liu, T. et al., 2007 ISTH meeting,abstract #P-M-035; Henrik, A. et al., 2011 ISTH meeting, abstract#P=MO-181; Liu, T. et al., 2011 ISTH meeting abstract #P-WE-131.

Plasma von Willebrand Factor (VWF) has a half-life of approximately 12hours (ranging from 9 to 15 hours).http://www.nhlbi.nih.gov/guidelines/vwd/2_scientificoverview.htm (lastvisited Oct. 22, 2011). The VWF half-life may be affected by a number offactors: glycosylation pattern, ADAMTS-13 (a disintegrin andmetalloprotease with thrombospondin motif-13), and various mutations inVWF.

In plasma, 95-98% of FVIII circulates in a tight non-covalent complexwith full-length VWF. The formation of this complex is important for themaintenance of appropriate plasma levels of FVIII in vivo. Lenting etal., Blood. 92(11): 3983-96 (1998); Lenting et al., J. Thromb. Haemost.5(7): 1353-60 (2007). The full-length wild-type FVIII is mostly presentas a heterodimer having a heavy chain (MW 200 kd) and a light chain (MW73 kd). When FVIII is activated due to proteolysis at positions 372 and740 in the heavy chain and at position 1689 in the light chain, the VWFbound to FVIII is removed from the activated FVIII. The activated FVIII,together with activated factor IX, calcium, and phospholipid (“tenasecomplex”), involves in the activation of factor X, generating largeamounts of thrombin. Thrombin, in turn, then cleaves fibrinogen to formsoluble fibrin monomers, which then spontaneously polymerize to form thesoluble fibrin polymer. Thrombin also activates factor XIII, which,together with calcium, serves to crosslink and stabilize the solublefibrin polymer, forming cross-linked (insoluble) fibrin. The activatedFVIII is cleared fast from the circulation by proteolysis.

Due to the frequent dosing and inconvenience caused by the dosingschedule, there is still a need to develop FVIII products requiring lessfrequent administration, i.e., a FVIII product that has a half-lifelonger than the 1.5 to 2 fold half-life limitation.

BRIEF SUMMARY OF THE INVENTION

The present invention is drawn to a chimeric protein comprising a FactorVIII (“FVIII”) protein and an adjunct moiety (“AM”), wherein the adjunctmoiety inhibits or prevents endogenous VWF from binding to the FVIIIprotein. The FVIII protein and the adjunct moiety are linked to eachother by a covalent bond in order to prevent dissociation of the adjunctmoiety in the presence of endogenous VWF. In one embodiment, thecovalent bond is a peptide bond, a disulfide bond, or a linker, which isstrong enough to prevent dissociation of the adjunct moiety from theFVIII protein in the presence of endogenous VWF. In another embodiment,the adjunct moiety prevents the FVIII protein from being cleared througha VWF clearance pathway. In other embodiments, the adjunct moietyinhibits or prevents endogenous VWF from binding to the FVIII protein byshielding or blocking a VWF binding site on the FVIII protein. Forexample, VWF binding site is located in the A3 domain or the C2 domainof the FVIII protein or both the A3 domain and the C2 domain.

In some embodiments, the chimeric protein includes a constructcomprising a FVIII protein and an adjunct moiety linked to each other bya covalent bond, wherein the chimeric protein does not comprise a FVIIIhalf-life limiting factor, which induces a half-life limitation of theFVIII protein, e.g., a full-length VWF protein or a mature VWF protein.Therefore, in some embodiments, the half-life of the FVIII protein ofthe chimeric protein is extendable beyond the half-life limitation ofthe FVIII protein in the presence of endogenous VWF.

In certain embodiments, the adjunct moiety has at least one VWF-likeFVIII protecting property. Examples of the VWF-like FVIII protectingproperty include, but are not limited to, protecting the FVIII proteinfrom one or more protease cleavages, protecting the FVIII protein fromactivation, stabilizing the heavy chain and/or the light chain of theFVIII protein, or preventing clearance of the FVIII protein by one ormore scavenger receptors. In one embodiment, the adjunct moietycomprises a polypeptide, a non-polypeptide moiety, or both. In anotherembodiment, the adjunct moiety can be a polypeptide comprising an aminoacid sequence of at least about 40, at least about 50, at least about60, at least about 70, at least about 80, at least about 90, at leastabout 100, at least about 110, at least about 120, at least about 130,at least about 140, at least about 150, at least about 200, at leastabout 250, at least about 300, at least about 350, at least about 400,at least about 450, at least about 500, at least about 550, at leastabout 600, at least about 650, at least about 700, at least about 750,at least about 800, at least about 850, at least about 900, at leastabout 950, or at least about 1000 amino acids in length. In certainembodiments, the adjunct moiety comprises a VWF fragment, animmunoglobulin constant region or a portion thereof, albumin or afragment thereof, an albumin binding moiety, a PAS sequence, a HAPsequence, transferrin or a fragment thereof, or any combinationsthereof. In other embodiments, the adjunct moiety is a non-polypeptidemoiety comprising polyethylene glycol (PEG), polysialic acid,hydroxyethyl starch (HES), a derivative thereof, or any combinationsthereof.

In certain embodiments, the adjunct moiety comprises a VWF fragmentcomprising a D′ domain and a D3 domain of VWF, wherein the VWF fragmentis associated with the FVIII protein by a non-covalent bond in additionto the covalent bond between the FVIII protein and the adjunct moiety(VWF fragment). In one example, the VWF fragment is a monomer. Inanother example, the VWF fragment comprises two, three, four, five, orsix VWF fragments linked to one or more of each other.

In one aspect, the chimeric protein comprises an adjunct moiety, e.g., aVWF fragment, and at least one heterologous moiety (H1) and an optionallinker between the adjunct moiety, e.g., VWF fragment, and theheterologous moiety (H1). In one embodiment, the heterologous moiety(H1) can comprise a moiety that extends the half-life of the FVIIIprotein, e.g., a polypeptide selected from the group consisting of animmunoglobulin constant region or a portion thereof, albumin or afragment thereof, an albumin binding moiety, a PAS sequence, a HAPsequence, transferrin or a fragment thereof, and any combinationsthereof or a non-polypeptide moiety selected from the group consistingof polyethylene glycol (PEG), polysialic acid, hydroxyethyl starch(HES), a derivative thereof, and any combinations thereof. In oneembodiment, the heterologous moiety (H1) comprises a first Fc region. Inanother embodiment, the heterologous moiety (H1) comprises an amino acidsequence comprising at least about 50 amino acids, at least about 100amino acids, at least about 150 amino acids, at least about 200 aminoacids, at least about 250 amino acids, at least about 300 amino acids,at least about 350 amino acids, at least about 400 amino acids, at leastabout 450 amino acids, at least about 500 amino acids, at least about550 amino acids, at least about 600 amino acids, at least about 650amino acids, at least about 700 amino acids, at least about 750 aminoacids, at least about 800 amino acids, at least about 850 amino acids,at least about 900 amino acids, at least about 950 amino acids, or atleast about 1000 amino acids. In other embodiments, the chimeric proteincomprises a linker between the adjunct moiety, e.g., a VWF fragment, andthe heterologous moiety (H1), which is a cleavable linker.

In another aspect, the FVIII protein in the chimeric protein comprisesFVIII and at least one heterologous moiety (H2). In one embodiment, theheterologous moiety (H2) is capable of extending the half-life of theFVIII protein, e.g., a polypeptide selected from the group consisting ofan immunoglobulin constant region or a portion thereof, albumin or afragment thereof, an albumin binding moiety, a PAS sequence, a HAPsequence, transferrin or a fragment thereof, and any combinationsthereof or a non-polypeptide moiety comprising polyethylene glycol(PEG), polysialic acid, hydroxyethyl starch (HES), a derivative thereof,and any combinations thereof. In a particular embodiment, theheterologous moiety (H2) comprises a second Fc region.

In some embodiments, the chimeric protein comprises a first polypeptidechain comprising the VWF fragment, a first heterologous moiety, and alinker and a second polypeptide chain comprising the FVIII protein and asecond heterologous moiety, wherein the first polypeptide chain and thesecond polypeptide chain are linked to each other by a covalent bond. Inone example, the first heterologous moiety and the second heterologousmoiety are linked to each other by the covalent bond, e.g., a disulfidebond, a peptide bond, or a linker, wherein the covalent bond preventsreplacement of the VWF fragment in the first polypeptide chain withendogenous VWF in vivo. In some embodiments, the linker between theFVIII protein and the second heterologous moiety is a cleavable linker.

In certain embodiments, the first heterologous moiety (H1) linked to theVWF fragment and the second heterologous moiety (H2) linked to the FVIIIprotein are linked by a linker, e.g., a scFc linker, which is aprocessable linker.

In yet other embodiments, the FVIII protein in the chimeric proteinfurther comprises a third heterologous moiety (H3), a fourthheterologous moiety (H4), a fifth heterologous moiety (H5), a sixthheterologous moiety (H6), or any combinations thereof. In oneembodiment, one or more of the third heterologous moiety (H3), thefourth heterologous moiety (H4), the fifth heterologous moiety (H5), thesixth heterologous moiety (H6) are capable of extending the half-life ofthe FVIII protein. In another embodiments, the third heterologous moiety(H3), the fourth heterologous moiety (H4), the fifth heterologous moiety(H5), and the sixth heterologous moiety (H6) are linked to the Cterminus or N terminus of FVIII or inserted between two amino acids ofFVIII. In other embodiments, one or more of the third heterologousmoiety (H3), the fourth heterologous moiety (H4), the fifth heterologousmoiety (H5), or the sixth heterologous moiety (H6) comprises an aminoacid sequence comprising at least about 50 amino acids, at least about100 amino acids, at least about 150 amino acids, at least about 200amino acids, at least about 250 amino acids, at least about 300 aminoacids, at least about 350 amino acids, at least about 400 amino acids,at least about 450 amino acids, at least about 500 amino acids, at leastabout 550 amino acids, at least about 600 amino acids, at least about650 amino acids, at least about 700 amino acids, at least about 750amino acids, at least about 800 amino acids, at least about 850 aminoacids, at least about 900 amino acids, at least about 950 amino acids,or at least about 1000 amino acids.

In some embodiments, the linker between the FVIII protein and the secondheterologous moiety or the linker between the VWF fragment and the firstheterologous moiety further comprises a first cleavage site (P1) at theN-terminal region of the linker, a second cleavage site (P2) at theC-terminal region of the linker, or both. In other embodiments, one ormore of the linker between the FVIII protein and the adjunct moiety, thelinker between the FVIII protein and the second heterologous moiety, andthe linker between the VWF fragment and the first heterologous moietyhave a length of about 1 to about 2000 amino acids.

In other embodiments, the chimeric protein comprises a FVIII protein andan adjunct moiety, which are linked by a linker between the FVIIIprotein and the adjunct moiety, wherein the linker further comprises asortase recognition motif, e.g., the sequence of LPXTG (SEQ ID NO: 106).

The present invention is directed to a von Willebrand Factor (VWF)fragment comprising the D′ domain and the D3 domain of VWF, wherein theVWF fragment binds to Factor VIII (FVIII) and inhibits binding ofendogenous VWF to a FVIII protein. In one embodiment, the VWF fragmentof the invention is not amino acids 764 to 1274 of SEQ ID NO: 2. In oneembodiment, the FVIII protein, without the VWF fragment, has a half-lifecomparable to wild-type FVIII. In another embodiment, the FVIII proteinis a fusion protein comprising FVIII and a heterologous moiety that iscapable of extending half-life of FVIII. The heterologous moiety can bea polypeptide, a non-polypeptide moiety, or both. The heterologouspolypeptide moiety can be selected from the group consisting of animmunoglobulin constant region or a portion thereof, albumin or afragment thereof, an albumin binding moiety, a PAS sequence, a HAPsequence, transferrin or a fragment thereof, and any combinationthereof. In other embodiments, the heterologous moiety is animmunoglobulin constant region or a portion thereof, e.g., an Fc region.In still other embodiments, the non-polypeptide moiety is selected fromthe group consisting of polyethylene glycol (PEG), polysialic acid,hydroxyethyl starch (HES), a derivative thereof, and any combinationsthereof. In certain embodiments, The FVIII protein comprises a firstpolypeptide chain and a second polypeptide chain, wherein the firstpolypeptide chain comprises FVIII and a first Fc region and the secondpolypeptide chain comprises a second Fc region without FVIII.

In another embodiment, the VWF fragment extends a half-life of FVIII.The amino acid sequence of the D′ domain can be at least 90%, 95%, 96%,97%, 98%, 99%, or 100% identical to amino acids 764 to 866 of SEQ ID NO:2. Also, the amino acid sequence of the D3 domain can be at least 90%,95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 867 to 1240 ofSEQ ID NO: 2. In certain embodiments, the VWF fragment contains at leastone amino acid substitution at a residue corresponding to residue 1099,residue 1142, or both of SEQ ID NO: 2. In a particular embodiment, a VWFfragment comprises, consisting essentially of, or consists of aminoacids 764 to 1240 of SEQ ID NO: 2. The VWF fragment can further comprisethe D1 domain, the D2 domain, or the D1 and D2 domains of VWF. In someembodiments, the VWF fragment further comprises a VWF domain selectedfrom the group consisting of the A1 domain, the A2 domain, the A3domain, the D4 domain, the B1 domain, the B2 domain, the B3 domain, theC1 domain, the C2 domain, the CK domain, one or more fragments thereof,and any combinations thereof. In other embodiments, the VWF fragment ispegylated, glycosylated, hesylated, or polysialylated.

The present invention is also directed to a chimeric protein comprisinga VWF fragment described herein, a heterologous moiety, and an optionallinker between the VWF fragment and the heterologous moiety. Theheterologous moiety can be a polypeptide, a non-polypeptide moiety, orboth. In one embodiment, the heterologous polypeptide moiety is selectedfrom the group consisting of an immunoglobulin constant region or aportion thereof, albumin or a fragment thereof, an albumin bindingmoiety, a PAS sequence, a HAP sequence, transferrin or a fragmentthereof, and any combination thereof. In another embodiment, theheterologous non-polypeptide moiety is selected from group consisting ofpolyethylene glycol (PEG), polysialic acid, hydroxyethyl starch (HES), aderivative thereof, and any combinations thereof. In a particularembodiment, the heterologous moiety is a first Fc region. The chimericprotein can further comprise a second Fc region, wherein the second Fcregion is linked to or associated with the first Fc region or linked toor associated with the VWF fragment.

In one aspect, a chimeric protein of the invention comprises a formulaselected from the group consisting of:

(aa) V-L1-H1-L2-H2, (bb) H2-L2-H1-L1-V, (cc) H1-L1-V-L2-H2, and (dd)H2-L2-V-L 1-H1,

wherein the V is one or more of the VWF fragments described herein,

each of L1 and L2 is an optional linker;

H1 is a first heterologous moiety;

(-) is a peptide bond or one or more amino acids; and

H2 is an optional second heterologous moiety.

In one embodiment, H1 is a first heterologous moiety, e.g., a half-lifeextending molecule which is known in the art. In one embodiment, thefirst heterologous moiety is a polypeptide. The first heterologouspolypeptide moiety is selected from the group consisting of animmunoglobulin constant region or a portion thereof, albumin or afragment thereof, an albumin binding moiety, a PAS sequence, a HAPsequence, transferrin or a fragment thereof, and any combinationsthereof. In another embodiment, H1 is a non-polypeptide moiety selectedfrom the group consisting of polyethylene glycol (PEG), polysialic acid,hydroxyethyl starch (HES), a derivative thereof, and any combinationsthereof. H2 is an optional second heterologous moiety, e.g., a half-lifeextending molecule which is known in the art. In one embodiment, thesecond heterologous moiety can be selected from the group consisting ofan immunoglobulin constant region or a portion thereof, albumin or afragment thereof, an albumin binding moiety, a PAS sequence, a HAPsequence, transferrin or a fragment thereof, and any combinationthereof. In another embodiment, H2 is a non-polypeptide moiety, which isselected from the group consisting of polyethylene glycol (PEG),polysialic acid, hydroxyethyl starch (HES), a derivative thereof, andany combinations thereof. In certain embodiments, H1 is a first Fcregion and H2 is a second Fc region. The first Fc region and the secondFc region can be the same or different and can be linked to each otherby a linker or a covalent bond, e.g., a disulfide bond. In anotherembodiment, the second Fc region is linked to or associated with aFactor VIII protein. Optionally, there could be a third heterologousmoiety, H3, which is a half-life extender, which is linked to the VWFfragment, the first heterologous moiety, or the second heterologousmoiety. Non-limiting examples of the third heterologous moiety caninclude a polypeptide or a non-polypeptide moiety or both. In oneembodiment, the third heterologous polypeptide moiety can be selectedfrom the group consisting of an immunoglobulin constant region or aportion thereof, albumin or a fragment thereof, an albumin bindingmoiety, a PAS sequence, a HAP sequence, transferrin or a fragmentthereof, or any combinations thereof. In another embodiment, H2 is anon-polypeptide moiety, which is be selected from the group consistingof polyethylene glycol (PEG), polysialic acid, hydroxyethyl starch(HES), a derivative thereof, and any combinations thereof. In someembodiments, H3 is linked to the VWF fragment or the first or the secondheterologous moiety by a cleavable linker, e.g., a thrombin cleavablelinker. Non-limiting examples of the linkers are disclosed elsewhereherein.

In another aspect, the invention provides a chimeric protein comprisinga VWF fragment described herein, a FVIII protein, and an optional linkerbetween the VWF fragment and the FVIII protein. The VWF fragment can bebound to the FVIII protein. In one embodiment, a chimeric proteincomprises a VWF fragment described herein, which is linked to aheterologous moiety. The heterologous moiety can be a moiety thatextends the half-life of the protein, which comprises a polypeptide, anon-polypeptide moiety, or both. Examples of such a heterologouspolypeptide moiety include, e.g., an immunoglobulin constant region or aportion thereof, albumin or a fragment thereof, an albumin bindingmoiety, a PAS sequence, a HAP sequence, any derivatives or variantsthereof, or any combinations thereof. Examples of a non-polypeptidemoiety include, e.g., polyethylene glycol (PEG), polysialic acid,hydroxyethyl starch (HES), a derivative thereof, or any combinationsthereof. In another embodiment, the heterologous moiety is a first Fcregion linked to the VWF fragment. In other embodiments, the chimericprotein further comprises a second Fc region linked to the FVIIIprotein. The VWF fragment or the FVIII protein can be linked to thefirst Fc region or the second Fc region, respectively, by a linker. Instill other embodiments, a chimeric protein comprises a VWF fragmentdescribed herein linked to a first heterologous moiety, e.g., first Fcregion, and a FVIII protein linked to a second heterologous moiety,e.g., second Fc region, wherein the VWF fragment is further linked tothe second heterologous moiety (e.g., second Fc region) or the FVIIIprotein by a linker or by covalent bond or the first heterologous moiety(e.g., Fc region) is further linked to the FVIII protein or the secondheterologous moiety (e.g., second Fc region) by a linker or a covalentbond. In some embodiments, the FVIII of the chimeric protein has apartial B-domain. In some embodiments, the FVIII protein with a partialB-domain is FVIII198 (SEQ ID NO: 105). In other embodiments, thechimeric protein further comprises a sortase recognition motif.

In some embodiments, as a result of the invention the half-life of theFVIII protein is extended compared to a FVIII protein without the VWFfragment or wildtype FVIII. The half-life of the FVIII protein is atleast about 1.5 times, at least about 2 times, at least about 2.5 times,at least about 3 times, at least about 4 times, at least about 5 times,at least about 6 times, at least about 7 times, at least about 8 times,at least about 9 times, at least about 10 times, at least about 11times, or at least about 12 times longer than the half-life of a FVIIIprotein without the VWF fragment. In one embodiment, the half-life ofFVIII is about 1.5-fold to about 20-fold, about 1.5 fold to about 15fold, or about 1.5 fold to about 10 fold longer than the half-life ofwild-type FVIII. In another embodiment, the half-life of the FVIII isextended about 2-fold to about 10-fold, about 2-fold to about 9-fold,about 2-fold to about 8-fold, about 2-fold to about 7-fold, about 2-foldto about 6-fold, about 2-fold to about 5-fold, about 2-fold to about4-fold, about 2-fold to about 3-fold, about 2.5-fold to about 10-fold,about 2.5-fold to about 9-fold, about 2.5-fold to about 8-fold, about2.5-fold to about 7-fold, about 2.5-fold to about 6-fold, about 2.5-foldto about 5-fold, about 2.5-fold to about 4-fold, about 2.5-fold to about3-fold, about 3-fold to about 10-fold, about 3-fold to about 9-fold,about 3-fold to about 8-fold, about 3-fold to about 7-fold, about 3-foldto about 6-fold, about 3-fold to about 5-fold, about 3-fold to about4-fold, about 4-fold to about 6 fold, about 5-fold to about 7-fold, orabout 6-fold to about 8 fold as compared to wild-type FVIII or a FVIIIprotein without the VWF fragment. In other embodiments, the half-life ofFVIII is at least about 17 hours, at least about 18 hours, at leastabout 19 hours, at least about 20 hours, at least about 21 hours, atleast about 22 hours, at least about 23 hours, at least about 24 hours,at least about 25 hours, at least about 26 hours, at least about 27hours, at least about 28 hours, at least about 29 hours, at least about30 hours, at least about 31 hours, at least about 32 hours, at leastabout 33 hours, at least about 34 hours, at least about 35 hours, atleast about 36 hours, at least about 48 hours, at least about 60 hours,at least about 72 hours, at least about 84 hours, at least about 96hours, or at least about 108 hours. In still other embodiments, thehalf-life of FVIII is about 15 hours to about two weeks, about 16 hoursto about one week, about 17 hours to about one week, about 18 hours toabout one week, about 19 hours to about one week, about 20 hours toabout one week, about 21 hours to about one week, about 22 hours toabout one week, about 23 hours to about one week, about 24 hours toabout one week, about 36 hours to about one week, about 48 hours toabout one week, about 60 hours to about one week, about 24 hours toabout six days, about 24 hours to about five days, about 24 hours toabout four days, about 24 hours to about three days, or about 24 hoursto about two days.

In some embodiments, the average half-life of the FVIII protein persubject is about 15 hours, about 16 hours, about 17 hours, about 18hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours,about 23 hours, about 24 hours (1 day), about 25 hours, about 26 hours,about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours,about 36 hours, about 40 hours, about 44 hours, about 48 hours (2 days),about 54 hours, about 60 hours, about 72 hours (3 days), about 84 hours,about 96 hours (4 days), about 108 hours, about 120 hours (5 days),about six days, about seven days (one week), about eight days, aboutnine days, about 10 days, about 11 days, about 12 days, about 13 days,or about 14 days.

In another aspect, a chimeric protein of the invention comprises aformula selected from the group consisting of:

(a) V-L1-H1-L3- C-L2-H2, (b) H2-L2-C-L3- H1-L1-V, (c) C-L2-H2- L3-V-L1-H1, (d) H1-L1-V-L3-H2-L2-C, (e) H1-L1-V-L3-C-L2-H2, (f) H2-L2-C-L3-V-L1-H1, (g) V-L1-H1-L3- H2-L2-C, (h) C-L2-H2- L3- H1-L1-V, (i)H2-L3-H1-L1-V-L2-C, (j) C-L2-V-L1-H1-L3-H2, (k) V-L2-C-L1-H1-L3-H2, and(l) H2-L3-H1-L1-C-L2-V,

wherein V is a VWF fragment described herein;

each of L1 or L2, is an optional linker, e.g., a thrombin cleavablelinker;

L3 is an optional linker, e.g., scFc linker, e.g., a processable linker;

each of H1 or H2 is an optional heterologous moiety; and

C is a FVIII protein; and

(-) is a peptide bond or one or more amino acids.

In other aspects, a chimeric protein of the invention comprises aformula selected from the group consisting of:

(m) V-L1-H1: H2-L2-C, (n) V-L1-H1:C-L2-H2, (o) H1-L1-V:H2-L2-C, (p)H1-L1-V:C-L2-H2, (q) V:C-L1-H1:H2, (r) V:H1-L1-C:H2, (s) H2:H1-L1-C:V,(t) C:V-L1-H1:H2, and (u) C:H1-L1-V:H2,

wherein V is a VWF fragment described herein;

each of L1 or L2, is an optional linker, e.g., a thrombin cleavablelinker;

each of H1 or H2 is an optional heterologous moiety; and

C is a FVIII protein;

(-) is a peptide bond or one or more amino acids; and

(:) is a chemical or physical association between H1 and H2, between Vand C, and between V and H1 and C and H2. (:) represents a chemicalassociation, e.g., at least one non-peptide bond. In certainembodiments, the chemical association, i.e., (:) is a covalent bond. Insome embodiments, the association between H1 and H2 is a covalent bond,e.g., a disulfide bond. In other embodiments, the chemical association,i.e., (:) is a non-covalent interaction, e.g., an ionic interaction, ahydrophobic interaction, a hydrophilic interaction, a Van der Waalsinteraction, a hydrogen bond. In certain embodiments, the associationbetween the FVIII protein and the VWF fragment is a non-covalent bond.In other embodiments, (:) is a non-peptide covalent bond. In still otherembodiments, (:) is a peptide bond. In one embodiment, H1 is a firstheterologous moiety. In one embodiment, the first heterologous moiety iscapable of extending half-life of the FVIII activity. In anotherembodiment, the first heterologous moiety is a polypeptide, anon-polypeptide moiety, or both. In one embodiment, the firstheterologous polypeptide moiety can be selected from the groupconsisting of an immunoglobulin constant region or a portion thereof,albumin or fragment thereof, an albumin binding moiety, a PAS sequence,a HAP sequence, transferrin or a fragment thereof, and any combinationsthereof. In another embodiment, the non-polypeptide moiety is selectedfrom the group consisting of polyethylene glycol (PEG), polysialic acid,hydroxyethyl starch (HES), a derivative thereof, and any combinationsthereof. In some embodiments, H2 is a second heterologous moiety. Thesecond heterologous moiety can also be a half-life extender known in theart and can be a polypeptide, a non-polypeptide moiety, or a combinationof both. In one embodiment, the second heterologous moiety is selectedfrom the group consisting of an immunoglobulin constant region or aportion thereof, albumin or fragment thereof, an albumin binding moiety,a PAS sequence, a HAP sequence, transferrin or a fragment thereof, andany combinations thereof. In certain embodiments, the non-polypeptidemoiety is selected from the group consisting of polyethylene glycol(PEG), polysialic acid, hydroxyethyl starch (HES), a derivative thereof,and any combinations thereof. In a particular embodiment, H1 is a firstFc region. In some embodiments, H2 is a second Fc region. Optionally,there could be a third heterologous moiety, H3, which is a half-lifeextender. H3 can be linked to one or more of V, C, H1, or H2 by anoptional linker, e.g., a cleavable linker, e.g., a thrombin cleavablelinker. Non-limiting examples of the third heterologous moiety caninclude an immunoglobulin constant region or a portion thereof, albuminor a fragment thereof, polyethylene glycol (PEG), a PAS sequence, andhydroxyethyl starch (HES) or a derivative thereof.

In certain embodiments, one or more of the linkers used to connect theVWF fragment, the FVIII protein, the first heterologous moiety, and/orthe second heterologous moiety of formulas (a) to (u) to each other is acleavable linker. One or more of the cleavage sites used in the chimericprotein can be cleaved by a protease selected from the group consistingof factor XIa, factor XIIa, kallikrein, factor VIIa, factor IXa, factorXa, factor Ha (thrombin), Elastase-2, Granzyme-B, TEV, Enterokinase,Protease 3C, Sortase A, MMP-12, MMP-13, MMP-17, and MMP-20. In otherembodiments, one or more linkers used in formulas (a) to (l) (e.g., L3)comprise a processable linker. The processable linkers can be cleaved byan intracellular enzyme upon secretion. The processable linker cancomprise a first cleavage site (P1) at the N-terminal region of thelinker, a second cleavage site (P2) at the C-terminal region of thelinker, or both.

In some embodiments, one or more of the linkers used in the inventionhave a length of at least about 1 to 2000 amino acids. In a specificembodiment, one or more of the linkers used in the invention have alength of at least about 20, 35, 42, 48, 73, 98, 144, 288, 324, 576, or864 amino acids. In a particular embodiment, one or more of the linkerscomprise a gly/ser peptide. The gly/ser peptide can be (Gly4 Ser)₃ or(Gly4 Ser)₄.

In other aspects, a FVIII protein in a chimeric protein is a functionalFactor VIII protein. The FVIII protein can comprise one or more domainsof FVIII selected from the group consisting of the A1 domain, the A2domain, the B domain, the A3 domain, the C1 domain, the C2 domain, oneor more fragment thereof, and any combinations thereof. In oneembodiment, the FVIII protein comprises the B domain or a portionthereof. In another embodiment, the FVIII protein is SQ B domain deletedFVIII. In other embodiments, the FVIII protein comprises single chainFVIII. In still other embodiments, the FVIII protein comprises a heavychain of FVIII and a light chain of Factor VIII, wherein the heavy chainand the light chain are associated with each other by a metal bond. Incertain embodiments, the FVIII protein has a low affinity to or does notbind to a low-density lipoprotein receptor-related protein (LRP). Forexample, a FVIII protein useful for the invention can contain at leastone amino acid substitution that lowers the affinity to or eliminatesthe binding to the LRP. Non-limiting examples of the at least one aminoacid substitution is at a residue corresponding to residue 471, residue484, residue 487, residue 490, residue 497, residue 2092, residue 2093or two or more combinations thereof of full-length mature FVIII. In someembodiments, the FVIII protein in a chimeric protein of this inventioncontains at least one amino acid substitution, which induces the FVIIIprotein to be more stable than a FVIII protein without the substitution.In other embodiments, the FVIII protein contains at least one amino acidsubstitution in the A2 domain and at least one amino acid substitutionin the A3 domain, wherein the A2 domain and the A3 domain are associatedto each other by a covalent bond. Non-limiting examples of the aminoacid substitution in the A2 domain is at a residue corresponding residue662 or 664 of full-length mature FVIII. In addition, non-limitingexamples of the amino acid substitution in the A3 domain is at a residuecorresponding to residue 1826 or 1828 of full-length mature FVIII ispolysialylated.

In further aspects, the invention provides a polynucleotide encoding aVWF fragment described herein or a chimeric protein described herein, ora set of polynucleotides comprising a first nucleotide chain and asecond nucleotide chain, wherein the first nucleotide chain encodes theVWF fragment and the second nucleotide chain encodes the second Fcregion or the clotting factor or fragment thereof of the chimericprotein. In one embodiment, the set of polynucleotides further comprisesa third polynucleotide chain, which encodes a proprotein convertasebelongs to the subtilisin-like proprotein convertase family.Non-limiting examples of the proprotein convertase include proproteinconvertase subtilisin/kexin type 3 (PACE or PCSK3), proproteinconvertase subtilisin/kexin type 5 (PCSK5 or PC5), proprotein convertasesubtilisin/kexin type 7 (PCSK7 or PC7), or a yeast Kex 2. In still otheraspects, the invention includes a vector comprising the polynucleotideor the set of polynucleotides and one or more promoters operably linkedto the polynucleotide or the set of polynucleotides or a set of vectorscomprising a first vector and a second vector, wherein the first vectorencodes the first polynucleotide chain of the set of polynucleotides andthe second vector encodes the second polynucleotide chain of the set ofpolynucleotides. The set of vectors can further comprise a third vector,which comprises a third polynucleotide chain encoding PC5 or PC7. Insome embodiments, the vector further comprises PACE. In someembodiments, PACE cleaves the D1D2 domains of the VWF fragment.

In some aspects, the invention is directed to a pharmaceuticalcomposition comprising the VWF fragment, the chimeric protein, thepolynucleotide, the set of polynucleotides, the vector, or the set ofvectors, and a pharmaceutically acceptable carrier. The composition ofthis invention can extend the half-life of Factor VIII. In otheraspects, the invention includes a host cell comprising thepolynucleotide, the set of polynucleotides, the vector, or the sets ofvectors.

In other aspects, the present invention is drawn to a chimeric proteincomprising a FVIII protein, an adjunct moiety and an optional linker,wherein the adjunct moiety inhibits or prevents endogenous VWF frombinding to the FVIII protein and has at least one VWF-like FVIIIprotecting property. The VWF-like FVIII protecting property comprisesprotecting the FVIII protein from one or more protease cleavages,protecting the FVIII protein from activation, stabilizing the heavychain and/or the light chain of the FVIII protein, or preventingclearance of the FVIII protein by one or more scavenger receptors.

The adjunct moiety in the chimeric protein can inhibit or preventendogenous VWF from binding to the FVIII protein by shielding orblocking a VWF binding site on the FVIII protein. In some embodiments,the VWF binding site is located in the A3 domain or the C2 domain of theFVIII protein or both A3 domain and C2 domain of the FVIII protein. Inanother embodiment, the VWF binding site is the amino acid sequencecorresponding to amino acids 1669 to 1689 and 2303 to 2332 of SEQ ID NO:16. In some embodiments, the adjunct moiety is a polypeptide, anon-polypeptide moiety, or both. The polypeptide useful as the adjunctmoiety can comprise an amino acid sequence of at least 40, 50, 60, 70,80, 90, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 450, 500,550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 amino acids inlength. For example, the polypeptide useful as an adjunct moiety can beselected from the group consisting of a VWF fragment, an immunoglobulinconstant region or a portion thereof, albumin or a fragment thereof, analbumin binding moiety, a PAS sequence, a HAP sequence, other half-lifeextending technologies, and any combinations thereof. Thenon-polypeptide moiety useful as an adjunct moiety can be selected fromthe group consisting of polyethylene glycol (PEG), polysialic acid,hydroxyethyl starch (HES) or a derivative thereof, and any combinationsthereof. In one embodiment, the adjunct moiety is the VWF fragmentdescribed herein. The adjunct moiety and the FVIII protein can belinked, e.g., by a linker, or associated with each other. The linker cancomprise a cleavable linker, e.g., a thrombin cleavable linker.

In one aspect, the invention provides a method of preventing orinhibiting binding of a FVIII protein with endogenous VWF comprisingadding an effective amount of the VWF fragment, the chimeric protein,the polynucleotide, or the set of polynucleotides to a cell comprising aFVIII protein or a polynucleotide encoding the FVIII protein, whereinthe VWF fragment binds to the FVIII protein. In another aspect, theinvention includes a method of preventing or inhibiting binding of theFVIII protein with endogenous VWF comprising adding an effective amountof the chimeric protein, the polynucleotide, or the set ofpolynucleotides to a subject in need thereof, wherein the VWF fragmentbinds to the FVIII protein and thus prevents or inhibits binding of theFVIII protein. In some aspects, the invention includes a method ofextending or increasing half-life of a FVIII protein, wherein the methodcomprises adding an effective amount of the VWF fragment, the chimericprotein, the polynucleotide, or the set of polynucleotides to a cellcomprising a FVIII protein or a polynucleotide encoding the FVIIIprotein or to a subject in need thereof, wherein the VWF fragment bindsto the FVIII protein. In other aspects, the invention is drawn to amethod of preventing or inhibiting clearance of a FVIII protein from acell, wherein the method comprises adding an effective amount of the VWFfragment, the chimeric protein, the polynucleotide, or the set ofpolynucleotides to a cell comprising a FVIII protein or a polynucleotideencoding the FVIII protein or to a subject in need thereof, wherein theVWF fragment binds to the FVIII protein.

In another aspect, the invention is directed to a method of treating ableeding disease or disorder in a subject in need thereof comprisingadministering an effective amount of the VWF fragment, the chimericprotein, the polynucleotide, or the set of polynucleotides, wherein thebleeding disease or disorder is selected from the group consisting of ableeding coagulation disorder, hemarthrosis, muscle bleed, oral bleed,hemorrhage, hemorrhage into muscles, oral hemorrhage, trauma, traumacapitis, gastrointestinal bleeding, intracranial hemorrhage,intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture,central nervous system bleeding, bleeding in the retropharyngeal space,bleeding in the retroperitoneal space, and bleeding in the illiopsoassheath. In other embodiments, the treatment is prophylactic oron-demand. In still other embodiments, the invention is a method oftreating a disease or disorder associated with Type 2N von Willebrand'sdisease to a subject in need thereof, comprising administering aneffective amount of the VWF fragment, the chimeric protein, thepolynucleotide, or the set of polynucleotides, wherein the disease ordisorder is treated.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1A-F. Schematic diagrams of VWF proteins. FIG. 1A shows two VWFfragments containing amino acids 1 to 276 of SEQ ID NO: 73 (amino acids764 to 1039 of SEQ ID NO: 2). VWF-001 is synthesized without thepre/propeptide sequences of VWF, while VWF-009 is synthesized with thepre/propeptide sequences (D1 and D2 domains). The prepeptide of VWF-009is cleaved during synthesis, and VWF-009 contains the propeptide withthe D′ and D3 domain sequences. FIG. 1B shows three VWF fragmentscontaining amino acids 1 to 477 of SEQ ID NO: 73 (amino acids 764 to1240 of SEQ ID NO: 2). VWF-002 is synthesized without the pre/propeptidesequences. VWF-010 contains the D1D2 domains in addition to the D′D3domains. VWF-013 contains the D1D2D′D3 domains in addition to alanineresidues substituting cysteines at residues 336 and 379 of SEQ ID NO:72. FIG. 1C shows two VWF fragments containing the D′D3 domains and aportion of the A1 domain. VWF-003 has amino acids 764 to 1274 of SEQ IDNO: 2). VWF-011 contains the D1D2 domains in addition to the D′D3domains. FIG. 1D shows two constructs, VWF-004 and VWF-012. VWF-004contains the D′D3 domains and the complete sequence of A1 domain.VWF-012 contains the D1D2D′D3 domains and the complete sequence of A1domain. FIG. 1E shows three constructs. VWF-006 contains the D1D2D′D3domains and the CK domain of VWF (cysteine knot domain). VWF-008 is thefull-length VWF. VWF-031 (VWF-Fc) shows a construct containing theD1D2D′D3 domains linked to a single Fc region by a cleavable linker.VWF-053 is the D1D2 domains. FIG. 1F shows full-length VWF proteincomprising propeptide (the D1 and D2 domains) and mature subunits (theD′, D3, A1, A2, A3, D4, B1-3, C1-2 domains). The VWF protein is about250 kDa protein and forms multimers (>20 MDa) by disulfide bonding. TheVWF protein associates with FVIII (95-98%) in non-covalent complex andthen extends half-life of FVIII by protecting FVIII from proteasecleavage/activation, stabilizing heavy & light chain, and preventingclearance of FVIII by scavenger receptors. The VWF protein also canlimit half-life of FVIII by clearance of FVIII-VWF complex through VWFreceptors and preventing pinocytosis and recycling of rFVIIIFc.

FIGS. 2A-2B. Schematic diagrams of examples of VWF:FVIII heterodimerconstructs. The left construct shows a VWF fragment having the D′D3domains of full-length VWF (amino acids 1-477 of SEQ ID NO: 73) andcontaining alanine substitutions at residues 336 and 379 of SEQ ID NO:72. The chimeric protein construct (FVIII 064/065) comprises theC-terminus of a VWF fragment linked to a first Fc region by a linker andFVIII is linked to a second Fc region, wherein the second Fc region isfurther linked to the N-terminus of a VWF fragment by a linker (e.g.,formula C-H1-L1-V-L2-H2, wherein V is a VWF fragment, C is FVIII, H1 andH2 are Fc regions, and L1 and L2 are cleavable linkers). The constructin FIG. 2 b is an intracellularly processed VWF:FVIII heterodimerconstruct where the linker between the second Fc and the N-terminus ofthe VWF fragment has been cleaved. FVIII-064 contains the D′D3 domainsof VWF (amino acids 1 to 477 of SEQ ID NO: 73 with C336A and C379substitutions). FVIII-065 contains the D′D3 domains of VWF (amino acids1 to 276 of SEQ ID NO: 73). FVIII-136 contains FVIIIFc linked to theD′D3 fragment-Fc by a linker that can be processed by an intracellularprotease enzyme. When FVIII-136 is expressed, the enzyme cleaves thelinker between the second Fc (fused to FVIII-LC) and the VWF D′D3fragment (fused to the first Fc), while the Fc region fused to (orlinked to) FVIII-LC forms a covalent bond (e.g., a disulfide bond) withthe first Fc fused to (or linked to) the VWF fragment. FVIII-148 issingle chain FVIIIFc with the D′D3 fragment (a single chain FVIII byintroducing R1645A/R1648A mutation into FVIII gene).

FIG. 3 . Schematic diagrams of examples of VWF:FVIII heterodimerconstructs containing examples of variable linkers between VWF and Fc.The constructs (FVIII-064, FVIII-159, FVIII-160, FVIII-178, andFVIII-179) have the common structure represented as formulaC-H1-L1-V-L2-H2, but contain examples of different linkers or amino acidsubstitutions. The constructs shown contain the same VWF fragment, whichis the D′ and D3 domains of VWF (i.e., amino acids 1 to 477 of SEQ IDNO: 73 with amino acid substitutions C336A and C379A). Construct FVIII64 has a thrombin cleavable linker (i.e., L2) between the VWF fragmentand the Fc (i.e., H2), which has 20 amino acids. Construct FVIII 159 hasa thrombin cleavable linker (i.e., L2) between the VWF fragment and theFc (i.e., H2), which has 35 amino acids. Construct FVIII 160 has athrombin cleavable linker (i.e., L2) between the VWF fragment and the Fc(i.e., H2), which has 48 amino acids. Constructs FVIII-180, FVIII-181,and FVIII-182 are derivatives of FVIII-160 containing K2092A mutation inFVIII C1 domain, K2093A mutation in FVIII C1 domain, and K2092A/K2093Amutations in FVIII C1 domain, respectively. Construct FVIII-178 has athrombin cleavable linker (i.e., L2) between the VWF fragment and the Fc(i.e., H2), which has 73 amino acids. Construct FVIII-179 has a thrombincleavable linker (i.e., L2) between the VWF fragment and the Fc (i.e.,H2), which has 98 amino acids.

FIGS. 4A-4H: Schematic diagrams of examples of FVIII-VWF constructs, inwhich VWF is D1D2D′D3 fragment of VWF, the Linker is a variable lengthlinker containing a cleavage site, e.g., a thrombin cleavage site, SCFVIII is a single chain FVIII, which contains the R1645A/R1648Asubstitutions, H is a heterologous moiety, e.g., an immunoglobulinconstant region or a portion thereof, a moiety for conjugatingpolyethylene glycol (PEG) and/or PEG, an albumin or albumin fragment, analbumin binding moiety, a HAP sequence, a moiety for polysialylationand/or polysialic acid, a moiety for hydroxyethyl starch (HES) and/orHES, or a PAS sequence, etc., HC FVIII is a heavy chain of FVIII, LCFVIII is a light chain of FVIII, and Fc is an Fc region of animmunoglobulin constant region. FIG. 4A has a formula of VWF-Linker-SCFVIII. FIG. 4B has a formula of VWF-Linker-H-Linker-SC FVIII. Thelinkers (the first linker between VWF and H and the second linkerbetween H and SC FVIII) can be identical or different. FIG. 4C has aformula of VWF-Linker-SC FVIII-Linker-H. The linkers (the first linkerbetween VWF and SC FVIII and the second linker between SC FVIII and H)can be identical or different. FIG. 4D has a formula of VWF-Linker-HCFVIII-H-Linker-LC FVIII. The linkers (the first linker between VWF andHC FVIII and the second linker between H and LC FVIII) can be identicalor different. FIG. 4E has a formula of HC FVIII-H-LC FVIII-Linker-firstFc-Linker-VWF-Linker-second Fc. The linkers (the first linker between LCFVIII and first Fc, the second linker between first Fc and VWF, and thethird linker between VWF and second Fc) can be identical or different.The linkers can be a cleavable linker. For example, the linker betweenfirst Fc and VWF can be a cleavable linker comprising a cleavage site atthe N-terminus and/or the C-terminus of the linker. The first Fc and thesecond Fc can be identical or different. FIG. 4F has a formula of HCFVIII-H-LC FVIII-Linker-first Fc-Linker-VWF-Linker-second Fc. Thelinkers (the first linker between LC FVIII and first Fc, the secondlinker between first Fc and VWF, and the third linker between VWF andsecond Fc) can be identical or different. One or more linkers can be acleavable linker. For example, the linker between the first Fc and VWFcan be a cleavable linker comprising a cleavage site at the N-terminusand/or the C-terminus of the linker. The first Fc and the second Fc canbe identical or different. FIG. 4G has a formula of SCFVIII-Linker-Fc-Linker-VWF-H-Linker-Fc. FIG. 4H has a formula ofPegylated or Hesylated SC FVIII-Linker-Fc-Linker-VWF-H-Linker-Fc. Thelinkers (the first linker between SC FVIII and first Fc, the secondlinker between first Fc and VWF, and the third linker between H andsecond Fc) can be identical or different. One or more linkers can be acleavable linker. For example, the linker between the first Fc and VWFcan be a cleavable linker comprising a cleavage site at the N-terminusand/or the C-terminus of the linker. The first Fc and the second Fc canbe identical or different.

FIG. 5 . Schematic diagrams of FVIII-VWF heterodimer co-transfectionsystem. Construct FVIII-155 contains the full-length FVIII sequence(with an alanine residue substituting the arginine residues at 1645 and1648) linked to an Fc region. VWF-031 contains the D1D2D′D3 fragment(with an alanine residue substituting the Cysteine residues at 336 and379) which is linked to another Fc region with a 48 thrombin cleavablelinker. After intracellular processing, construct FVIII-155 produces afull length single chain FVIII (SCFVIII) fused to one Fc fragment,construct VWF-031 produces a 477 amino acids D′D3 fragment linked toanother Fc fragment. Two covalent bonds can be formed between the Fcfragments that are linked to the SC FVIII or the D′D3 fragment, this inturn allows a covalent association of FVIII and D′D3, which is the maincharacter of the desired final product.

FIG. 6 is the non-reducing and reducing SDS PAGE of VWF-009 (D1D2D′D31-276 aa×6 HIS), which shows VWF-009 exists as a monomer. Unprocessedmeans VVF-009 with the propeptide (the D1D2 domains).

FIG. 7 is the non-reducing and reducing SDS PAGE of VWF-002 (D′D3 1-477aa×6 his) or VWF-010 (D1D2D′D3 1-477 aa×6 his), which shows VWF-002exists as a monomer and VWF-010 exists as a dimer.

FIG. 8 shows thrombin digestion of FVIII-VWF heterodimer shown in FIG.2(b). Lane 1 shows marker. Lane 2 is rFVIII-Fc without thrombin. Lane 3is rFVIII-Fc with thrombin. Lane 5 is FVIIIFc-VWF. Lane 6 showsFVIIIFc-VWF and thrombin. A1 indicates A1 domain of FVIII, A2 indicatesA2 domain of FVIII, and Δa3 LC indicates the light chain of FVIII.

FIG. 9A-B shows the FVIII activity measured by a FVIII chromogenicassay. FIG. 9A shows pharmacokinetic profile of rFVIII and rFVIIIFc inHemA mouse. FIG. 9B shows PK profile of rFVIII and rFVIIIFc in FVIII/VWFDouble knockout (DKO) mouse. The Y axis shows FVIII activity in mIU/mL,and the X axis shows time.

FIG. 10A-B shows FVIII protection by the D′D3 fragments as shown bymFVIII plasma level (mIU/mL) and VWF expression level (nM/mL) 48 hourspost plasmid injection. The VWF fragments used to show FVIII protectionare VWF-001 (276aa, monomer), VWF-009 (276aa, monomer), VWF-002 (477aa,monomer), VWF-010 (477aa,dimer), VWF-003 (511aa, monomer), VWF-011(511aa, dimer), VWF-004 (716aa, monomer), VWF-012 (716aa, dimer),VWF-006, and VWF-008.

FIGS. 11A-11B show the pharmacokinetic profile of rBDD-FVIII inFVIII-VWF DKO mice when co-administered with D′D3 fragments. FIG. 11Ashows FVIII activity (mIU/mL) measured by a FVIII chromogenic assayafter co-administration of rBDD-FVIII and VWF-002 or rBDD-FVIII andVWF-010 or rBDD-FVIII alone in FVIII/VWF DKO mice. FIG. 11B showsVWF-002 and VWF-010 plasma level (ng/mL) after administration. The Xaxis represents time in hours.

FIGS. 12A-12C show pharmacokinetic profile of rFVIIIFc in VWF D′D3expressing mice. FIG. 12A shows the timeline of hydrodynamic injection(HDI) of the D′D3 domain encoding plasmid DNA (day −5), intravenousdosing of rFVIIIFc (day 0), and PK sample collection (day0-day3). FIG.12B shows post rFVIIIFc infusion plasma FVIII activity (mIU/mL) measuredby a FVIII chromogenic assay in FVIII/VWF DKO mice with HDI of theD1D2D′D3 domains (477aa) (circle) and the D1D2D′D3 domains (477aa) withcysteine substitutions (rectangle) in FVIII/VWF DKO mice. The FVIIIactivity in control mice without HDI of the D′D3 domains is shown astriangle. FIG. 12C show the D′D3 plasma level (ng/mL) after HDIadministration of the D1D2D′D3 dimer or the D1D2D′D3 monomer DNAconstruct. The X axis represents time in hours.

FIG. 13 shows D′D3-Fc linker selection by HDI in FVIII/VWF DKO mice.Different lengths of the linkers (20aa (FVIII-064), 35aa (FVIII-159), or48aa (FVIII-160)) were inserted between the D′D3 domains and the Fcregion. The FVIII activity (mIU/ml) was measured by a FVIII chromogenicassay after HDI in FVIII/VWF DKO mice.

FIG. 14 shows HDI of Single Chain FVIIIFc/D′D3 heterodimer in FVIII/VWFDKO mice. The FVIII activities of processed (dual chain) rFVIIIFc-D′D3(pSYN-FVIII-136) and Single Chain rFVIIIFc-D′D3 (pSYN-FVIII-148) weremeasured 24 hours and 48 hours after HDI.

FIG. 15 shows binding affinity of FVIII-155/VWF-031 heterodimer toimmobilized hVWF by Octet assay. FVIIIFc, FVIII, and IgG were also usedas controls. The x-axis shows time in seconds, and the y-axis shows thebinding in nanometer (nm).

FIG. 16 shows FVIII-155/VWF-031 pharmacokinetics in FVIII/VWF deficient(FVIII/VWF DKO) mice. The x-axis indicates time in hours, and the y-axisindicates FVIII recovery v. input in percent.

FIGS. 17A-17C: Schematic diagrams of examples of VWF fragmentconstructs, in which VWF is D1D2D′D3 fragment of VWF; the Linker is avariable length linker containing a cleavage site, e.g., a thrombincleavage site; H is a heterologous moiety, e.g., an immunoglobulinconstant region or a portion thereof, a moiety for conjugatingpolyethylene glycol (PEG) and/or PEG, an albumin or albumin fragment, analbumin binding moiety, a HAP sequence, a moiety for polysialylationand/or polysialic acid, a moiety for hydroxyethyl starch (HES) and/orHES, or a PAS sequence, etc.; and Fc is an Fc region of animmunoglobulin. FIG. 17A has a formula of D1D2-D′partial D3-H-PartialD3-Linker-Fc. FIG. 17B has a formula of D1D2-Partial D′-H-partialD′D3-Linker-Fc. FIG. 17C has a formula of D1D2-Pegylated or HesylatedD′D3- Linker-Fc. The linker can be optionally cleaved.

FIGS. 18A-18B: FIG. 18A shows FVIIIFc loses FVIII activity in both HemA(diamond) and DKO (square) plasma over time. FVIII activity is measuredby chromogenic assay. X-axis shows time in hours, and y-axis showsrelative activity. FIG. 18B shows that the loss in FVIII activity is dueto the dissociation or degradation of the heavy chain (HC). The leftpanel shows an immuno-precipitation assay using sheep anti-FVIIIpolyclonal antibody in Bio-rad 4-15% gel. The gel was reduced and imagedby Bio-rad system. Lane 1 shows Bio-rad unstain marker; lane 2 showsFVIIIFc and PBS; lane 3 shows FVIIIFc and DKO plasma; and lane 5 showssheep anti-FVIII polyclonal antibody alone. The right panel showsWestern analysis of the gel using FVIII anti-heavy chain antibody(GMA012). Lane 1 shows Bio-rad unstain marker; lane 2 shows FVIIIFc andPBS; lane 3 shows FVIIIFc and DKO plasma; and lane 4 shows sheepanti-FVIII polyclonal antibody alone.

FIG. 19 : shows FVIII activity of wild type FVIIIFc (circle), scFVIIIFc(single chain FVIII) (filled triangle), or FVIII:VWF heterodimer (e.g.,FVIII155/VWF31) (empty triangle) by chromogenic assay in DKO mouseplasma (left panel) and HemA mouse plasma (right panel) as a function oftime. Y axis shows relative FVIII activity. Wild type FVIIIFc containsdual chain of FVIII (i.e., FVIII heavy chain and FVIII light chain heldtogether non-covalently) and thus has three chains, a FVIII heavy chain,a FVIII light chain fused to an Fc, and an Fc alone. ScFVIIIFc containsa FVIII single chain and thus has two chains, one with a single chainFVIII fused to an Fc and another with an Fc alone. The FVIII:VWFheterodimer (e.g., FVIII155/VWF031) contains single chain FVIII fused toan Fc and a VWF fragment (D′D3) fused to an Fc.

FIG. 20 shows processing of D1D2 domain from VWF fragment (e.g., VWF-031(D1D2D′D3Fc)) by PC5 or PACE (Furin) at different concentrations. TheD1D2 processing is shown on a Bio-rad 4-15% gel at a reduced conditionby Bio-rad imager. Lane 1 shows VWF031 alone; lane 2 shows PC5 alone;lane 3 shows PACE alone; lane 4 shows VWF031 and PC5 at 2.5%; lane 5shows VWF031 and PC5 at 5%; lane 6 shows VWF031 and PC5 at 7.5%; lane 7shows VWF031 and PC5 at 10%; lane 8 shows VWF031 and PACE at 2.5%; lane9 shows VWF031 and PACE at 5%; lane 10 shows VWF031 at 7.5%; and lane 11shows VWF031 and PACE at 10%.

FIGS. 21A-21B: FIG. 21A shows that a binding assay of a FVIII:VWFheterodimer (e.g., FVIII-155/VWF-031) by ForteBio octet instrument. Forthe assay, full length VWF was captured by using APS sensor. The bindingof FVIIIFc and FVIII to the full-length VWF is shown at the lower leftpanel. The lack of binding of FVIIIY1680 (a mutant having no affinityfor VWF) and FVIII:VWF heterodimer (FVIII155/VWF031) is shown at thelower right panel. FIG. 21B shows another binding assay of a FVIII:VWFheterodimer (e.g., FVIII-155/VWF-031). In this assay, the constructs(VWF031 construct, FVIII-155/VWF031, or FVIII) were immobilized onprotein G sensor. The binding of the constructs to FVIII was measured.

FIG. 22 shows binding affinity of VWF D′D3 domains with FVIII moleculemeasured by a surface plasma resonance experiment. The VWF031 construct(100 RU) was captured by 1000 RU anti-human IgG. B-domain deleted FVIIIwas applied in single cycle kinetics mode in 1:1 fit. The total numberwas 4.

FIG. 23 shows effects of different linker length in the FVIIIFc/VWFheterodimer constructs on pharmacokinetics when administered inFVIII/VWF DKO mice. Three different linkers (48 aa, 73aa, or 98aa) wereinserted between the D′D3 and the Fc, i.e., VWF031, VWF035, and VWF036.The FVIII activity normalized to 5 min value (%) is shown in Y-axis.

FIGS. 24A-24E show examples of sortase ligation of a VWF fragment withFVIII. FIG. 24A shows two ligation constructs, (1) a VWF fragment fusedto a sortase recognition motif (e.g., LPXTG) at the C-terminus and (2)FVIII having glycine (n) at the N-terminus. After reaction with sortase,the VWF fragment and the sortase recognition motif are ligated to theN-terminus of FVIII. FIG. 24B shows two ligation constructs, (1) FVIIIfused to a sortase recognition motif at its C-terminus and (2) a VWFfragment having glycine (n) at its N-terminus. After reaction withsortase, FVIII and the sortase recognition motif are fused to the VWFfragment at the N-terminus of the VWF fragment. FIG. 24C shows twoligation constructs, (1) a VWF fragment fused to a sortase recognitionmotif by a variable length linker and (2) FVIII fused to glycine (n) atits N-terminus. After reaction with sortase, the VWF fused by a linkerto the sortase recognition motif is ligated to the N-terminus of FVIII.FIG. 24D shows two ligation constructs, (1) FVIII fused by a variablelength linker to a sortase recognition motif and (2) a VWF fragmentfused to glycine (n) at its N-terminus. After reaction with sortase,FVIII fused by a linker to the sortase recognition motif is ligated tothe N terminus of VWF fragment. FIG. 24E shows a ligation constructcontaining a VWF fragment fused by a variable length linker to a sortaserecognition motif, which is also fused to a protease cleavage site(e.g., Thrombin cleavage site) fused by a variable length linker to anFc.

FIG. 25 shows a schematic comparison of FVIII155 and FVIII198. FVIII155encodes a single chain FVIIIFc protein. FVIII198 is a partial B-domaincontaining single chain FVIIIFc molecule-226N6. 226 represents theN-terminus 226 amino acid of the FVIII B-domain, and N6 represents sixN-glycosylation sites in the B-domain.

FIG. 26A shows a stability assay measuring the relativity activity ofFVIII155 and FVIII198 in DKO plasma as a function of time. As can beseen in the figure, the presence of the partial B-domain in FVIII198increased the stability of single chain FVIIIFc in comparison toFVIII155; FIG. 26B shows a comparison of the half-lives of FVIII198,FVIII155, and dual chain (dcFVIIIFc) in DKO mice. As can be seen in thefigure, single chain FVIII (FVIII155) has a 1.5 fold increase in halflife in comparison to dual chain FVIII. Single chain FVIII with the266N6 B-domain (FVIII198) had a further 1.5 fold increase in half life.The graph shows the FVIII recovery v. the 5 minute value (%) as afunction of time.

DETAILED DESCRIPTION OF THE INVENTION Definitions

It is to be noted that the term “a” or “an” entity refers to one or moreof that entity; for example, “a nucleotide sequence,” is understood torepresent one or more nucleotide sequences. As such, the terms “a” (or“an”), “one or more,” and “at least one” can be used interchangeablyherein.

The term “polynucleotide” or “nucleotide” is intended to encompass asingular nucleic acid as well as plural nucleic acids, and refers to anisolated nucleic acid molecule or construct, e.g., messenger RNA (mRNA)or plasmid DNA (pDNA). In certain embodiments, a polynucleotidecomprises a conventional phosphodiester bond or a non-conventional bond(e.g., an amide bond, such as found in peptide nucleic acids (PNA)). Theterm “nucleic acid” refers to any one or more nucleic acid segments,e.g., DNA or RNA fragments, present in a polynucleotide. By “isolated”nucleic acid or polynucleotide is intended a nucleic acid molecule, DNAor RNA, which has been removed from its native environment. For example,a recombinant polynucleotide encoding a Factor VIII polypeptidecontained in a vector is considered isolated for the purposes of thepresent invention. Further examples of an isolated polynucleotideinclude recombinant polynucleotides maintained in heterologous hostcells or purified (partially or substantially) from otherpolynucleotides in a solution. Isolated RNA molecules include in vivo orin vitro RNA transcripts of polynucleotides of the present invention.Isolated polynucleotides or nucleic acids according to the presentinvention further include such molecules produced synthetically. Inaddition, a polynucleotide or a nucleic acid can include regulatoryelements such as promoters, enhancers, ribosome binding sites, ortranscription termination signals.

As used herein, a “coding region” or “coding sequence” is a portion ofpolynucleotide which consists of codons translatable into amino acids.Although a “stop codon” (TAG, TGA, or TAA) is typically not translatedinto an amino acid, it may be considered to be part of a coding region,but any flanking sequences, for example promoters, ribosome bindingsites, transcriptional terminators, introns, and the like, are not partof a coding region. The boundaries of a coding region are typicallydetermined by a start codon at the 5′ terminus, encoding the aminoterminus of the resultant polypeptide, and a translation stop codon atthe 3′terminus, encoding the carboxyl terminus of the resultingpolypeptide. Two or more coding regions of the present invention can bepresent in a single polynucleotide construct, e.g., on a single vector,or in separate polynucleotide constructs, e.g., on separate (different)vectors. It follows, then, that a single vector can contain just asingle coding region, or comprise two or more coding regions, e.g., asingle vector can separately encode a binding domain-A and a bindingdomain-B as described below. In addition, a vector, polynucleotide, ornucleic acid of the invention can encode heterologous coding regions,either fused or unfused to a nucleic acid encoding a binding domain ofthe invention. Heterologous coding regions include without limitationspecialized elements or motifs, such as a secretory signal peptide or aheterologous functional domain.

Certain proteins secreted by mammalian cells are associated with asecretory signal peptide which is cleaved from the mature protein onceexport of the growing protein chain across the rough endoplasmicreticulum has been initiated. Those of ordinary skill in the art areaware that signal peptides are generally fused to the N-terminus of thepolypeptide, and are cleaved from the complete or “full-length”polypeptide to produce a secreted or “mature” form of the polypeptide.In certain embodiments, a native signal peptide, e.g., an immunoglobulinheavy chain or light chain signal peptide is used, or a functionalderivative of that sequence that retains the ability to direct thesecretion of the polypeptide that is operably associated with it.Alternatively, a heterologous mammalian signal peptide, e.g., a humantissue plasminogen activator (TPA) or mouse ß-glucuronidase signalpeptide, or a functional derivative thereof, can be used.

The term “downstream” refers to a nucleotide sequence that is located 3′to a reference nucleotide sequence. In certain embodiments, downstreamnucleotide sequences relate to sequences that follow the starting pointof transcription. For example, the translation initiation codon of agene is located downstream of the start site of transcription.

The term “upstream” refers to a nucleotide sequence that is located 5′to a reference nucleotide sequence. In certain embodiments, upstreamnucleotide sequences relate to sequences that are located on the 5′ sideof a coding region or starting point of transcription. For example, mostpromoters are located upstream of the start site of transcription.

As used herein, the term “regulatory region” refers to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding region, and whichinfluence the transcription, RNA processing, stability, or translationof the associated coding region. Regulatory regions may includepromoters, translation leader sequences, introns, polyadenylationrecognition sequences, RNA processing sites, effector binding sites andstem-loop structures. If a coding region is intended for expression in aeukaryotic cell, a polyadenylation signal and transcription terminationsequence will usually be located 3′ to the coding sequence.

A polynucleotide which encodes a gene product, e.g., a polypeptide, caninclude a promoter and/or other transcription or translation controlelements operably associated with one or more coding regions. In anoperable association a coding region for a gene product, e.g., apolypeptide, is associated with one or more regulatory regions in such away as to place expression of the gene product under the influence orcontrol of the regulatory region(s). For example, a coding region and apromoter are “operably associated” if induction of promoter functionresults in the transcription of mRNA encoding the gene product encodedby the coding region, and if the nature of the linkage between thepromoter and the coding region does not interfere with the ability ofthe promoter to direct the expression of the gene product or interferewith the ability of the DNA template to be transcribed. Othertranscription control elements, besides a promoter, for exampleenhancers, operators, repressors, and transcription termination signals,can also be operably associated with a coding region to direct geneproduct expression.

A variety of transcription control regions are known to those skilled inthe art. These include, without limitation, transcription controlregions which function in vertebrate cells, such as, but not limited to,promoter and enhancer segments from cytomegaloviruses (the immediateearly promoter, in conjunction with intron-A), simian virus 40 (theearly promoter), and retroviruses (such as Rous sarcoma virus). Othertranscription control regions include those derived from vertebrategenes such as actin, heat shock protein, bovine growth hormone andrabbit ß-globin, as well as other sequences capable of controlling geneexpression in eukaryotic cells. Additional suitable transcriptioncontrol regions include tissue-specific promoters and enhancers as wellas lymphokine-inducible promoters (e.g., promoters inducible byinterferons or interleukins).

Similarly, a variety of translation control elements are known to thoseof ordinary skill in the art. These include, but are not limited toribosome binding sites, translation initiation and termination codons,and elements derived from picornaviruses (particularly an internalribosome entry site, or IRES, also referred to as a CITE sequence).

The term “expression” as used herein refers to a process by which apolynucleotide produces a gene product, for example, an RNA or apolypeptide. It includes without limitation transcription of thepolynucleotide into messenger RNA (mRNA), transfer RNA (tRNA), smallhairpin RNA (shRNA), small interfering RNA (siRNA) or any other RNAproduct, and the translation of an mRNA into a polypeptide. Expressionproduces a “gene product.” As used herein, a gene product can be eithera nucleic acid, e.g., a messenger RNA produced by transcription of agene, or a polypeptide which is translated from a transcript. Geneproducts described herein further include nucleic acids with posttranscriptional modifications, e.g., polyadenylation or splicing, orpolypeptides with post translational modifications, e.g., methylation,glycosylation, the addition of lipids, association with other proteinsubunits, or proteolytic cleavage.

A “vector” refers to any vehicle for the cloning of and/or transfer of anucleic acid into a host cell. A vector may be a replicon to whichanother nucleic acid segment may be attached so as to bring about thereplication of the attached segment. A “replicon” refers to any geneticelement (e.g., plasmid, phage, cosmid, chromosome, virus) that functionsas an autonomous unit of replication in vivo, i.e., capable ofreplication under its own control. The term “vector” includes both viraland nonviral vehicles for introducing the nucleic acid into a cell invitro, ex vivo or in vivo. A large number of vectors are known and usedin the art including, for example, plasmids, modified eukaryoticviruses, or modified bacterial viruses. Insertion of a polynucleotideinto a suitable vector can be accomplished by ligating the appropriatepolynucleotide fragments into a chosen vector that has complementarycohesive termini.

Vectors may be engineered to encode selectable markers or reporters thatprovide for the selection or identification of cells that haveincorporated the vector. Expression of selectable markers or reportersallows identification and/or selection of host cells that incorporateand express other coding regions contained on the vector. Examples ofselectable marker genes known and used in the art include: genesproviding resistance to ampicillin, streptomycin, gentamycin, kanamycin,hygromycin, bialaphos herbicide, sulfonamide, and the like; and genesthat are used as phenotypic markers, i.e., anthocyanin regulatory genes,isopentanyl transferase gene, and the like. Examples of reporters knownand used in the art include: luciferase (Luc), green fluorescent protein(GFP), chloramphenicol acetyltransferase (CAT), -galactosidase (LacZ),-glucuronidase (Gus), and the like. Selectable markers may also beconsidered to be reporters.

The term “plasmid” refers to an extra-chromosomal element often carryinga gene that is not part of the central metabolism of the cell, andusually in the form of circular double-stranded DNA molecules. Suchelements may be autonomously replicating sequences, genome integratingsequences, phage or nucleotide sequences, linear, circular, orsupercoiled, of a single- or double-stranded DNA or RNA, derived fromany source, in which a number of nucleotide sequences have been joinedor recombined into a unique construction which is capable of introducinga promoter fragment and DNA sequence for a selected gene product alongwith appropriate 3′ untranslated sequence into a cell.

Eukaryotic viral vectors that can be used include, but are not limitedto, adenovirus vectors, retrovirus vectors, adeno-associated virusvectors, poxvirus, e.g., vaccinia virus vectors, baculovirus vectors, orherpesvirus vectors. Non-viral vectors include plasmids, liposomes,electrically charged lipids (cytofectins), DNA-protein complexes, andbiopolymers.

A “cloning vector” refers to a “replicon,” which is a unit length of anucleic acid that replicates sequentially and which comprises an originof replication, such as a plasmid, phage or cosmid, to which anothernucleic acid segment may be attached so as to bring about thereplication of the attached segment. Certain cloning vectors are capableof replication in one cell type, e.g., bacteria and expression inanother, e.g., eukaryotic cells. Cloning vectors typically comprise oneor more sequences that can be used for selection of cells comprising thevector and/or one or more multiple cloning sites for insertion ofnucleic acid sequences of interest.

The term “expression vector” refers to a vehicle designed to enable theexpression of an inserted nucleic acid sequence following insertion intoa host cell. The inserted nucleic acid sequence is placed in operableassociation with regulatory regions as described above.

Vectors are introduced into host cells by methods well known in the art,e.g., transfection, electroporation, microinjection, transduction, cellfusion, DEAE dextran, calcium phosphate precipitation, lipofection(lysosome fusion), use of a gene gun, or a DNA vector transporter.

“Culture,” “to culture” and “culturing,” as used herein, means toincubate cells under in vitro conditions that allow for cell growth ordivision or to maintain cells in a living state. “Cultured cells,” asused herein, means cells that are propagated in vitro.

As used herein, the term “polypeptide” is intended to encompass asingular “polypeptide” as well as plural “polypeptides,” and refers to amolecule composed of monomers (amino acids) linearly linked by amidebonds (also known as peptide bonds). The term “polypeptide” refers toany chain or chains of two or more amino acids, and does not refer to aspecific length of the product. Thus, peptides, dipeptides, tripeptides,oligopeptides, “protein,” “amino acid chain,” or any other term used torefer to a chain or chains of two or more amino acids, are includedwithin the definition of “polypeptide,” and the term “polypeptide” canbe used instead of, or interchangeably with any of these terms. The term“polypeptide” is also intended to refer to the products ofpost-expression modifications of the polypeptide, including withoutlimitation glycosylation, acetylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, or modification by non-naturally occurring amino acids. Apolypeptide can be derived from a natural biological source or producedrecombinant technology, but is not necessarily translated from adesignated nucleic acid sequence. It can be generated in any manner,including by chemical synthesis.

An “isolated” polypeptide or a fragment, variant, or derivative thereofrefers to a polypeptide that is not in its natural milieu. No particularlevel of purification is required. For example, an isolated polypeptidecan simply be removed from its native or natural environment.Recombinantly produced polypeptides and proteins expressed in host cellsare considered isolated for the purpose of the invention, as are nativeor recombinant polypeptides which have been separated, fractionated, orpartially or substantially purified by any suitable technique.

Also included in the present invention are fragments or variants ofpolypeptides, and any combination thereof. The term “fragment” or“variant” when referring to polypeptide binding domains or bindingmolecules of the present invention include any polypeptides which retainat least some of the properties (e.g., FcRn binding affinity for an FcRnbinding domain or Fc variant, coagulation activity for an FVIII variant,or FVIII binding activity for the VWF fragment) of the referencepolypeptide. Fragments of polypeptides include proteolytic fragments, aswell as deletion fragments, in addition to specific antibody fragmentsdiscussed elsewhere herein, but do not include the naturally occurringfull-length polypeptide (or mature polypeptide). Variants of polypeptidebinding domains or binding molecules of the present invention includefragments as described above, and also polypeptides with altered aminoacid sequences due to amino acid substitutions, deletions, orinsertions. Variants can be naturally or non-naturally occurring.Non-naturally occurring variants can be produced using art-knownmutagenesis techniques. Variant polypeptides can comprise conservativeor non-conservative amino acid substitutions, deletions or additions.

The term “VWF fragment” or “VWF fragments” used herein means any VWFfragments that interact with FVIII and retain at least one or moreproperties that are normally provided to FVIII by full-length VWF, e.g.,preventing premature activation to FVIIIa, preventing prematureproteolysis, preventing association with phospholipid membranes thatcould lead to premature clearance, preventing binding to FVIII clearancereceptors that can bind naked FVIII but not VWF-bound FVIII, and/orstabilizing the FVIII heavy chain and light chain interactions. The term“VWF fragment” as used herein does not include full length—or mature VWFprotein. In a particular embodiment, the “VWF fragment” as used hereincomprises a D′ domain and a D3 domain of the VWF protein, but does notinclude the A1 domain, the A2 domain, the A3 domain, the D4 domain, theB1 domain, the B2 domain, the B3 domain, the C1 domain, the C2 domain,and the CK domain of the VWF protein.

The term “half-life limiting factor” or “FVIII half-life limitingfactor” as used herein indicates a factor that prevents the half-life ofa FVIII protein from being longer than 1.5 fold or 2 fold compared towild-type FVIII (e.g., ADVATE® or REFACTO®). For example, full length ormature VWF can act as a FVIII half-life limiting factor by inducing theFVIII and VWF complex to be cleared from system by one or more VWFclearance pathways. In one example, endogenous VWF is a FVIII half-lifelimiting factor. In another example, a full-length recombinant VWFmolecule non-covalently bound to a FVIII protein is a FVIII-half-lifelimiting factor.

The term “endogenous VWF” as used herein indicates VWF moleculesnaturally present in plasma. The endogenous VWF molecule can bemultimer, but can be a monomer or a dimer. Endogenous VWF in plasmabinds to FVIII and forms a non-covalent complex with FVIII.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art, including basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Thus, if an amino acid in apolypeptide is replaced with another amino acid from the same side chainfamily, the substitution is considered to be conservative. In anotherembodiment, a string of amino acids can be conservatively replaced witha structurally similar string that differs in order and/or compositionof side chain family members.

As known in the art, “sequence identity” between two polypeptides isdetermined by comparing the amino acid sequence of one polypeptide tothe sequence of a second polypeptide. When discussed herein, whether anyparticular polypeptide is at least about 50%, 60%, 70%, 75%, 80%, 85%,90%, 95%, 99%, or 100% identical to another polypeptide can bedetermined using methods and computer programs/software known in the artsuch as, but not limited to, the BESTFIT program (Wisconsin SequenceAnalysis Package, Version 8 for Unix, Genetics Computer Group,University Research Park, 575 Science Drive, Madison, Wis. 53711).BESTFIT uses the local homology algorithm of Smith and Waterman,Advances in Applied Mathematics 2:482-489 (1981), to find the bestsegment of homology between two sequences. When using BESTFIT or anyother sequence alignment program to determine whether a particularsequence is, for example, 95% identical to a reference sequenceaccording to the present invention, the parameters are set, of course,such that the percentage of identity is calculated over the full-lengthof the reference polypeptide sequence and that gaps in homology of up to5% of the total number of amino acids in the reference sequence areallowed.

As used herein, an “amino acid corresponding to” or an “equivalent aminoacid” in a VWF sequence or a FVIII protein sequence is identified byalignment to maximize the identity or similarity between a first VWF orFVIII sequence and a second VWF or FVIII sequence. The number used toidentify an equivalent amino acid in a second VWF or FVIII sequence isbased on the number used to identify the corresponding amino acid in thefirst VWF or FVIII sequence.

A “fusion” or “chimeric” protein comprises a first amino acid sequencelinked to a second amino acid sequence with which it is not naturallylinked in nature. The amino acid sequences which normally exist inseparate proteins can be brought together in the fusion polypeptide, orthe amino acid sequences which normally exist in the same protein can beplaced in a new arrangement in the fusion polypeptide, e.g., fusion of aFactor VIII domain of the invention with an immunoglobulin Fc domain. Afusion protein is created, for example, by chemical synthesis, or bycreating and translating a polynucleotide in which the peptide regionsare encoded in the desired relationship. A chimeric protein can furthercomprises a second amino acid sequence associated with the first aminoacid sequence by a covalent, non-peptide bond or a non-covalent bond.

As used herein, the term “half-life” refers to a biological half-life ofa particular polypeptide in vivo. Half-life may be represented by thetime required for half the quantity administered to a subject to becleared from the circulation and/or other tissues in the animal. When aclearance curve of a given polypeptide is constructed as a function oftime, the curve is usually biphasic with a rapid α-phase and longerβ-phase. The α-phase typically represents an equilibration of theadministered Fc polypeptide between the intra- and extra-vascular spaceand is, in part, determined by the size of the polypeptide. The β-phasetypically represents the catabolism of the polypeptide in theintravascular space. In some embodiments, FVIII and chimeric proteinscomprising FVIII are monophasic, and thus do not have an alpha phase,but just the single beta phase. Therefore, in certain embodiments, theterm half-life as used herein refers to the half-life of the polypeptidein the β-phase. The typical β phase half-life of a human antibody inhumans is 21 days.

The term “heterologous” as applied to a polynucleotide or a polypeptide,means that the polynucleotide or polypeptide is derived from a distinctentity from that of the entity to which it is being compared. Therefore,a heterologous polypeptide linked to a VWF fragment means a polypeptidechain that is linked to a VWF fragment and is not a naturally occurringpart of the VWF fragment. For instance, a heterologous polynucleotide orantigen can be derived from a different species, different cell type ofan individual, or the same or different type of cell of distinctindividuals.

The term “linked” as used herein refers to a first amino acid sequenceor nucleotide sequence covalently or non-covalently joined to a secondamino acid sequence or nucleotide sequence, respectively. The term“covalently linked” or “covalent linkage” refers to a covalent bond,e.g., a disulfide bond, a peptide bond, or one or more amino acids,e.g., a linker, between the two moieties that are linked together. Thefirst amino acid or nucleotide sequence can be directly joined orjuxtaposed to the second amino acid or nucleotide sequence oralternatively an intervening sequence can covalently join the firstsequence to the second sequence. The term “linked” means not only afusion of a first amino acid sequence to a second amino acid sequence atthe C-terminus or the N-terminus, but also includes insertion of thewhole first amino acid sequence (or the second amino acid sequence) intoany two amino acids in the second amino acid sequence (or the firstamino acid sequence, respectively). In one embodiment, the first aminoacid sequence can be joined to a second amino acid sequence by a peptidebond or a linker. The first nucleotide sequence can be joined to asecond nucleotide sequence by a phosphodiester bond or a linker. Thelinker can be a peptide or a polypeptide (for polypeptide chains) or anucleotide or a nucleotide chain (for nucleotide chains) or any chemicalmoiety (for both polypeptide and polynucleotide chains). The covalentlinkage is sometimes indicated as (-) or hyphen.

As used herein the term “associated with” refers to a covalent ornon-covalent bond formed between a first amino acid chain and a secondamino acid chain. In one embodiment, the term “associated with” means acovalent, non-peptide bond or a non-covalent bond. In some embodimentsthis association is indicated by a colon, i.e., (:). In anotherembodiment, it means a covalent bond except a peptide bond. In otherembodiments, the term “covalently associated” as used herein means anassociation between two moieties by a covalent bond, e.g., a disulfidebond, a peptide bond, or one or more amino acids (e.g., a linker). Forexample, the amino acid cysteine comprises a thiol group that can form adisulfide bond or bridge with a thiol group on a second cysteineresidue. In most naturally occurring IgG molecules, the CH1 and CLregions are associated by a disulfide bond and the two heavy chains areassociated by two disulfide bonds at positions corresponding to 239 and242 using the Kabat numbering system (position 226 or 229, EU numberingsystem). Examples of covalent bonds include, but are not limited to, apeptide bond, a metal bond, a hydrogen bond, a disulfide bond, a sigmabond, a pi bond, a delta bond, a glycosidic bond, an agnostic bond, abent bond, a dipolar bond, a Pi backbond, a double bond, a triple bond,a quadruple bond, a quintuple bond, a sextuple bond, conjugation,hyperconjugation, aromaticity, hapticity, or antibonding. Non-limitingexamples of non-covalent bond include an ionic bond (e.g., cation-pibond or salt bond), a metal bond, an hydrogen bond (e.g., dihydrogenbond, dihydrogen complex, low-barrier hydrogen bond, or symmetrichydrogen bond), van der Walls force, London dispersion force, amechanical bond, a halogen bond, aurophilicity, intercalation, stacking,entropic force, or chemical polarity.

The term “monomer-dimer hybrid” used herein refers to a chimeric proteincomprising a first polypeptide chain and a second polypeptide chain,which are associated with each other by a disulfide bond, wherein thefirst chain comprises a clotting factor, e.g., Factor VIII, and an Fcregion and the second chain comprises, consists essentially of, orconsists of an Fc region without the clotting factor. The monomer-dimerhybrid construct thus is a hybrid comprising a monomer aspect havingonly one clotting factor and a dimer aspect having two Fc regions.

As used herein, the term “cleavage site” or “enzymatic cleavage site”refers to a site recognized by an enzyme. Certain enzymatic cleavagesites comprise an intracellular processing site. In one embodiment, apolypeptide has an enzymatic cleavage site cleaved by an enzyme that isactivated during the clotting cascade, such that cleavage of such sitesoccurs at the site of clot formation. Exemplary such sites include e.g.,those recognized by thrombin, Factor XIa or Factor Xa. Exemplary FXIacleavage sites include, e.g, TQSFNDFTR (SEQ ID NO: 47) and SVSQTSKLTR(SEQ ID NO: 48). Exemplary thrombin cleavage sites include, e.g,DFLAEGGGVR (SEQ ID NO: 49), TTKIKPR (SEQ ID NO: 50), LVPRG (SEQ ID NO:55) and ALRPR (amino acids 1 to 5 of SEQ ID NO: 51). Other enzymaticcleavage sites are known in the art.

As used herein, the term “processing site” or “intracellular processingsite” refers to a type of enzymatic cleavage site in a polypeptide whichis the target for enzymes that function after translation of thepolypeptide. In one embodiment, such enzymes function during transportfrom the Golgi lumen to the trans-Golgi compartment. Intracellularprocessing enzymes cleave polypeptides prior to secretion of the proteinfrom the cell. Examples of such processing sites include, e.g., thosetargeted by the PACE/furin (where PACE is an acronym for Paired basicAmino acid Cleaving Enzyme) family of endopeptidases. These enzymes arelocalized to the Golgi membrane and cleave proteins on the carboxyterminal side of the sequence motif Arg-[any residue]-(Lys or Arg)-Arg.As used herein the “furin” family of enzymes includes, e.g., PCSK1 (alsoknown as PC1/Pc3), PCSK2 (also known as PC2), PCSK3 (also known as furinor PACE), PCSK4 (also known as PC4), PCSK5 (also known as PC5 or PC6),PCSK6 (also known as PACE4), or PCSK7 (also known as PC7/LPC, PC8, orSPC7). Other processing sites are known in the art.

The term “Furin” refers to the enzymes corresponding to EC No.3.4.21.75. Furin is subtilisin-like proprotein convertase, which is alsoknown as PACE (Paired basic Amino acid Cleaving Enzyme). Furin deletessections of inactive precursor proteins to convert them intobiologically active proteins. During its intracellular transport,pro-peptide is cleaved from mature VWF molecule by a Furin enzyme in theGolgi.

In constructs that include more than one processing or cleavage site, itwill be understood that such sites may be the same or different.

Hemostatic disorder, as used herein, means a genetically inherited oracquired condition characterized by a tendency to hemorrhage, eitherspontaneously or as a result of trauma, due to an impaired ability orinability to form a fibrin clot. Examples of such disorders include thehemophilias. The three main forms are hemophilia A (factor VIIIdeficiency), hemophilia B (factor IX deficiency or “Christmas disease”)and hemophilia C (factor XI deficiency, mild bleeding tendency). Otherhemostatic disorders include, e.g., Von Willebrand disease, Factor XIdeficiency (PTA deficiency), Factor XII deficiency, deficiencies orstructural abnormalities in fibrinogen, prothrombin, Factor V, FactorVII, Factor X or factor XIII, Bernard-Soulier syndrome, which is adefect or deficiency in GPIb. GPIb, the receptor for VWF, can bedefective and lead to lack of primary clot formation (primaryhemostasis) and increased bleeding tendency), and thrombasthenia ofGlanzman and Naegeli (Glanzmann thrombasthenia). In liver failure (acuteand chronic forms), there is insufficient production of coagulationfactors by the liver; this may increase bleeding risk.

The chimeric molecules of the invention can be used prophylactically. Asused herein the term “prophylactic treatment” refers to theadministration of a molecule prior to a bleeding episode. In oneembodiment, the subject in need of a general hemostatic agent isundergoing, or is about to undergo, surgery. The chimeric protein of theinvention can be administered prior to or after surgery as aprophylactic. The chimeric protein of the invention can be administeredduring or after surgery to control an acute bleeding episode. Thesurgery can include, but is not limited to, liver transplantation, liverresection, dental procedures, or stem cell transplantation.

The chimeric protein of the invention is also used for on-demand (alsoreferred to as “episodic”) treatment. The term “on-demand treatment” or“episodic treatment” refers to the administration of a chimeric moleculein response to symptoms of a bleeding episode or before an activity thatmay cause bleeding. In one aspect, the on-demand (episodic) treatmentcan be given to a subject when bleeding starts, such as after an injury,or when bleeding is expected, such as before surgery. In another aspect,the on-demand treatment can be given prior to activities that increasethe risk of bleeding, such as contact sports.

As used herein the term “acute bleeding” refers to a bleeding episoderegardless of the underlying cause. For example, a subject may havetrauma, uremia, a hereditary bleeding disorder (e.g., factor VIIdeficiency) a platelet disorder, or resistance owing to the developmentof antibodies to clotting factors.

Treat, treatment, treating, as used herein refers to, e.g., thereduction in severity of a disease or condition; the reduction in theduration of a disease course; the amelioration of one or more symptomsassociated with a disease or condition; the provision of beneficialeffects to a subject with a disease or condition, without necessarilycuring the disease or condition, or the prophylaxis of one or moresymptoms associated with a disease or condition. In one embodiment, theterm “treating” or “treatment” means maintaining a FVIII trough level atleast about 1 IU/dL, 2 IU/dL, 3 IU/dL, 4 IU/dL, 5 IU/dL, 6 IU/dL, 7IU/dL, 8 IU/dL, 9 IU/dL, 10 IU/dL, 11 IU/dL, 12 IU/dL, 13 IU/dL, 14IU/dL, 15 IU/dL, 16 IU/dL, 17 IU/dL, 18 IU/dL, 19 IU/dL, or 20 IU/dL ina subject by administering a chimeric protein or a VWF fragment of theinvention. In another embodiment, treating or treatment meansmaintaining a FVIII trough level between about 1 and about 20 IU/dL,about 2 and about 20 IU/dL, about 3 and about 20 IU/dL, about 4 andabout 20 IU/dL, about 5 and about 20 IU/dL, about 6 and about 20 IU/dL,about 7 and about 20 IU/dL, about 8 and about 20 IU/dL, about 9 andabout 20 IU/dL, or about 10 and about 20 IU/dL. Treatment or treating ofa disease or condition can also include maintaining FVIII activity in asubject at a level comparable to at least about 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% ofthe FVIII activity in a non-hemophiliac subject. The minimum troughlevel required for treatment can be measured by one or more knownmethods and can be adjusted (increased or decreased) for each person.

Chimeric Proteins

The present invention is directed to extending the half-life of a FactorVIII protein by preventing or inhibiting a FVIII half-life limitingfactor (e.g. endogenous VWF) in vivo from associating with the FVIIIprotein. Endogenous VWF associates with about 95% to about 98% of FVIIIin non-covalent complexes. The endogenous VWFs bound to a FVIII proteinare known to protect FVIII in various ways. For example, full length VWF(as a multimer having about 250 kDa) can protect FVIII from proteasecleavage and FVIII activation, stabilize the FVIII heavy chain and/orlight chain, and prevent clearance of FVIII by scavenger receptors.However, at the same time, endogenous VWF limits the FVIII half-life bypreventing pinocytosis and by clearing FVIII-VWF complex from the systemthrough the VWF clearance pathway. It is believed, as shown in theexamples, that endogenous VWF is the half-life limiting factor thatprevents the half-life of a FVIII protein fused to a half-life extenderfrom being longer than about two-fold of wild-type FVIII. Therefore, thepresent invention prevents or inhibits interaction between endogenousVWF and a FVIII protein using an adjunct moiety, thereby preventing theFVIII protein from being cleared through the VWF clearance pathwayand/or inducing pinocytosis. In one embodiment, the adjunct moiety iscapable of preventing or inhibiting binding of the FVIII protein withendogenous VWF and has at least one VWF-like FVIII protecting property.In addition, the adjunct moiety reduces clearance of FVIII from thesystem by preventing or inhibiting interaction with endogenous VWF. Theadjunct moieties of the present invention bind to or are associated with(e.g., via non-covalent bonding) a FVIII protein and/or physically orchemically block the VWF binding site on the FVIII protein. The FVIIIprotein associated with the adjunct moiety is thus cleared from thecirculation more slowly by one or more VWF clearance receptors, ascompared to wild type FVIII or FVIII not associated with an adjunctmoiety.

Examples of the adjunct moieties of the present invention include, e.g.,polypeptides or chemical or physical modifications, additions,deletions, or variations of the FVIII protein. The adjunct moiety usefulin the present invention can comprise a polypeptide, a non-polypeptidemoiety, or both. Non-limiting examples of the polypeptide useful as anadjunct moiety include, e.g., a VWF fragment described herein, animmunoglobulin constant region or a portion thereof, transferrin or afragment thereof, albumin or a fragment thereof, an albumin bindingmoiety, a HAP sequence, a PAS sequence, or any combinations thereof.Non-limiting examples of the non-polypeptide moiety includespolyethylene glycol (PEG), polysialic acid, hydroxyethyl starch (HES), aderivative thereof, or any combination thereof. Other such moietiesuseful in present invention are known in the art.

In one embodiment, the adjunct moiety is associated (or linked) with theFVIII protein by a covalent or a non-covalent bond. In some instances,however, the physical blockage or chemical association (e.g.,non-covalent bonding) between the adjunct moiety and the FVIII proteinmay not be strong enough to provide a stable complex comprising theFVIII protein and the adjunct moiety in the presence of endogenous VWF.For example, a VWF fragment forming a non-covalent bond with a FVIIIprotein without any other connections may readily be dissociated in vivofrom the FVIII protein in the presence of endogenous VWF, replacing theVWF fragment (e.g., recombinant VWF, i.e., rVWF) with endogenous VWF.Therefore, the FVIII protein non-covalently bound to endogenous VWFwould undergo the VWF clearance pathway and be cleared from the system.In order to prevent the dissociation of the adjunct moiety with theFVIII protein, in some embodiments, the linkage between the FVIIIprotein and the adjunct moiety is a covalent bond, e.g., a peptide bond,one or more amino acids, or a disulfide bond. In certain embodiments,the association (i.e., linkage) between the adjunct moiety and the FVIIIprotein is a peptide bond or a linker between the FVIII protein and theadjunct moiety (“FVIII/AM linker”). Non-limiting examples of the linkeris described elsewhere herein. In some embodiments, the adjunct moietyis a polypeptide comprising, consisting essentially of, or consisting ofat least about 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000,1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000,or 4000 amino acids. In other embodiments, the adjunct moiety is apolypeptide comprising, consisting essentially of, or consisting ofabout 100 to about 200 amino acids, about 200 to about 300 amino acids,about 300 to about 400 amino acids, about 400 to about 500 amino acids,about 500 to about 600 amino acids, about 600 to about 700 amino acids,about 700 to about 800 amino acids, about 800 to about 900 amino acids,or about 900 to about 1000 amino acids. In some embodiments, the adjunctmoiety covalently associated with the FVIII protein is a VWF fragmentdescribed elsewhere herein.

In certain embodiments, the adjunct moiety chemically (e.g.,non-covalently) binds to or physically blocks one or more VWF bindingsites on a FVIII protein. The VWF binding site on a FVIII protein islocated within the A3 domain or the C2 domain of the FVIII protein. Instill other embodiments, the VWF binding site on a FVIII protein islocated within the A3 domain and C2 domain. For example, the VWF bindingsite on a FVIII protein can correspond to amino acids 1669 to 1689and/or 2303 to 2332 of SEQ ID NO: 16 [full-length mature FVIII].

In other embodiments, a chimeric protein of the invention comprises aFVIII protein linked to an adjunct moiety, wherein the adjunct moiety isa VWF molecule, e.g. a VWF fragment comprising a D′ domain and a D3domain, but not containing the VWF clearance receptor binding site, andshields or protects the VWF binding site on the FVIII protein, therebyinhibiting or preventing interaction of the FVIII protein withendogenous VWF. In certain embodiments, the adjunct moiety is a VWFfragment. The VWF fragment useful for the present invention contains theD′ domain and the D3 domain, still providing one or more advantages ofVWF-like property to the FVIII protein, but the VWF fragment does notundergo the VWF clearance pathway. The FVIII protein and the adjunctmoiety can be covalently associated by a linker (e.g., FVIII/AM linker).In one embodiment, the linker can be a cleavable linker. Non-limitingexamples of the linkers are disclosed elsewhere herein.

In still other embodiments, a chimeric protein of the inventioncomprises a FVIII protein and an immunoglobulin constant region or aportion thereof (i.e., an adjunct moiety), wherein the immunoglobulinconstant region or a portion thereof shields or protects the VWF bindingsite on the FVIII protein, thereby inhibiting or preventing interactionof the FVIII protein with endogenous VWF. In yet other embodiments, theimmunoglobulin constant region or a portion thereof is an Fc region.

In one aspect, the present invention is directed to a chimeric or fusionprotein or hybrid comprising one or more of the VWF fragments disclosedherein and uses of the same. The chimeric or fusion protein can be fusedor linked to one or more heterologous moiety (sometimes indicated hereinas H or H1). In one embodiment, the heterologous moiety (H1) is aheterologous peptide or a heterologous polypeptide that would notnaturally occur with and/or is linked to the VWF fragment. In anotherembodiment, the heterologous moiety (H1) is a non-polypeptide moiety,e.g., chemical modification or a combination of a peptide or polypeptideand a non-polypeptide moiety. In some embodiments, the VWF fragments arelinked or connected to the heterologous moiety (H1) by a linker (alsoreferred to herein as “VWF linker”). In one embodiment, the VWF linkeris a cleavable linker. Non-limiting examples of the linker between theVWF fragment and the heterologous moiety (H1) are disclosed elsewhereherein.

In one embodiment, the heterologous moiety (H1) useful in the inventionimproves one or more pharmacokinetic properties of the VWF fragmentswithout significantly affecting the VWF fragments' biological activityor function (e.g., its binding to or association with a FVIII protein).In another embodiment, the heterologous moiety (H1) linked to the VWFfragment can extend the half-life of the VWF fragments. Non-limitingexamples of the heterologous polypeptide moiety comprises animmunoglobulin constant region or a portion thereof, albumin or afragment thereof, an albumin binding moiety, a PAS sequence, a HAPsequence, transferrin or a fragment thereof, or two or more combinationsthereof. Non-limiting examples of the heterologous non-polypeptidemoiety include polyethylene glycol (PEG), polysialic acid, hydroxyethylstarch (HES), a derivative thereof, or any combinations thereof.

In some embodiments, a heterologous moiety (H1) can be used to connectthe VWF fragment and the FVIII protein by a covalent bond. Examples ofthe heterologous moiety that can provide the covalently linkage include,but are not limited to, an immunoglobulin constant region or a portionthereof comprising a hinge region, e.g., an Fc region or an FcRn bindingpartner. In a specific example, the FVIII protein is linked to a firstFc region, and the VWF fragment is linked to a second Fc region, whereinthe first Fc region and the second Fc region form one or more disulfidebond.

In some embodiments, the heterologous moiety (sometimes indicated hereinby “H” or “H1”) is an immunoglobulin constant region or a portionthereof. Non-limiting examples of the immunoglobulin constant region ora portion thereof can be selected from the group consisting of a CH1domain, a CH2 domain, a CH3 domain, a CH4 domain, a hinge domain, andtwo or more combinations thereof. In one embodiment, the immunoglobulinconstant region or a portion thereof comprises at least one CH1 domain,at least one CH2 domain, at least one CH3 domain, at least one CH4domain, or the functional fragments thereof. In another embodiment, theimmunoglobulin constant region or a portion thereof comprises at leastone hinge domain or a portion thereof and at least one CH2 domain or aportion thereof (e.g., in the hinge-CH2 orientation). In otherembodiments, the immunoglobulin constant domain or a portion thereofcomprises at least one CH2 domain or a portion thereof and at least oneCH3 domain or a portion thereof (e.g., in the CH2-CH3 orientation.)Examples of the combination include, but are not limited to, a CH2domain, a CH3 domain, and a hinge domain, which are also known as an Fcregion (or Fc domain), e.g., a first Fc region. In other embodiments,the heterologous moiety (H1) is linked to the VWF fragment by a linker.In certain embodiments, the heterologous moiety (H1) is an FcRn bindingpartner as described elsewhere herein. In other embodiments, theheterologous moiety (H1) is a hinge region.

In certain embodiments, the chimeric protein further comprises a second(or additional) heterologous moiety (sometimes indicated herein by“H2”). It is noted that the first heterologous moiety (H1) and thesecond heterologous moiety (H2) can be used interchangeably and can bethe same or different. The second heterologous moiety (H2) can be linkedto the FVIII protein or elsewhere in the chimeric protein by a peptidebond, one or more amino acids, or by a linker (e.g., FVIII linker iflinked to FVIII). Such constructs can sometimes be referred to asFVIII/VWF heterodimer. In one embodiment, the heterologous moiety (H2)comprises a heterologous polypeptide. In another embodiment, theheterologous moiety (H2) comprises a non-polypeptide moiety. In otherembodiments, the heterologous moiety (H2) comprises a combination of aheterologous moiety and a non-polypeptide moiety. The secondheterologous moiety (H2) can be a half-life extender. Non-limitingexamples of the second heterologous polypeptide moiety (H2) include animmunoglobulin constant region or a portion thereof, albumin or afragment thereof, an albumin binding moiety, a PAS sequence, a HAPsequence, transferrin or a fragment thereof, or two or more combinationsthereof. Non-limiting examples of the heterologous non-polypeptidemoiety include polyethylene glycol (PEG), polysialic acid, hydroxyethylstarch (HES), a derivative thereof, or any combinations thereof. Incertain embodiments, the first heterologous moiety (H1) and the secondheterologous moiety are the same or different. Either or both of thefirst heterologous moiety (H1) and the second heterologous moiety (H2)can confer half-life extension to the FVIII protein in a chimericprotein, provide a connection stronger than non-covalent association,i.e., by one or more covalent bonds between the FVIII protein and theVWF fragment in a chimeric protein, or both. Once the VWF fragment fusedor linked to the first heterologous moiety (H1) removes the half-lifeceiling by preventing or inhibiting interaction between the FVIIIprotein and the endogenous VWF protein, the FVIII protein fused to theheterologous moieties can reach to its full potential and can have ahalf-life of longer than two-fold compared to wild type FVIII.

In certain embodiments, the first heterologous moiety (e.g., a first Fcregion) linked to the VWF fragment and the second heterologous moiety(e.g., a second Fc region) linked to the FVIII protein are associatedwith each other such that the association prevents replacement of theVWF fragment by endogenous VWF in vivo. In one embodiment, the secondheterologous moiety is a second Fc region, wherein the second Fc regionis linked to or associated with the first heterologous moiety, e.g., thefirst Fc region, by a covalent bond, e.g., disulfide bond, a peptidebond, or a linker (one or more amino acids). For example, the secondheterologous moiety (e.g., the second Fc region) linked to the FVIIIprotein at one end can be further linked to the first heterologousmoiety (e.g., the first Fc region) linked to the VWF fragment by alinker (e.g., scFc linker) or associated with the first heterologousmoiety by a covalent or non-covalent bond. In another embodiment, thesecond heterologous moiety (e.g., the second Fc region) is linked to theVWF fragment that is already linked to first heterologous moiety. Insome embodiments, the chimeric protein comprises a first polypeptidechain comprising a VWF fragment and a first heterologous moiety and asecond polypeptide chain comprising a FVIII protein and a secondheterologous moiety, wherein the first polypeptide chain and the secondpolypeptide chain are associated, wherein the association between thefirst polypeptide chain comprising the first heterologous moiety and thesecond polypeptide chain comprising the second heterologous moiety is acovalent bond, thus allowing the VWF fragment and the FVIII proteinmaintain its interaction with each other. At the same time, endogenousVWF, which can form a non-covalent bond with the FVIII protein cannotreplace the covalently linked polypeptide chain comprising the VWFfragment.

The linker between the first heterologous moiety (H1) and the VWFfragment (e.g., VWF linker) can be a cleavable linker, e.g., a thrombincleavable linker. The cleavable linkers can be cleaved by a proteaseselected from the group consisting of factor XIa, factor XIIa,kallikrein, factor VIIa, factor IXa, factor Xa, factor IIa (thrombin),Elastase-2, Granzyme-B, TEV, Enterokinase, Protease 3C, Sortase A,MMP-12, MMP-13, MMP-17, MMP-20, and any combinations thereof. Thesecleavable linkers allow the VWF fragment to be cleaved and dissociatedfrom the FVIII protein upon activation of the clotting cascade,resulting in a FVIII protein with full activity potential.

In other embodiments, the chimeric protein is produced as a singlepolypeptide chain comprising a VWF fragment, a cleavable linker, a firstheterologous moiety (H1), a processable linker, a FVIII protein, and asecond heterologous moiety (H2) in any order. After synthesis, theprocessable linker can be cleaved by an intracellular protease enzymebefore secretion, thus making two polypeptide chains as described above.In the single chain construct before secretion, the second heterologousmoiety (e.g., the second Fc region) can be linked to the VWF fragment bya processable linker. In certain embodiments, one or more linkers cancomprise one or more cleavage sites.

In some embodiments, the chimeric protein of the invention furthercomprises a third heterologous moiety (sometimes indicated herein by“H3”). The third heterologous moiety (H3) can be a half-life extender.The heterologous moiety (H3) can comprise a heterologous polypeptide, anon-polypeptide moiety, or a combination of both. Non-limiting examplesof the third heterologous moiety (H3) include an immunoglobulin constantregion or a portion thereof, albumin or a fragment thereof, an albuminbinding moiety, a PAS sequence, a HAP sequence, transferrin or afragment thereof, any derivatives or variants thereof, or two or morecombinations thereof. Non-limiting examples of the non-polypeptidemoiety include polyethylene glycol (PEG), polysialic acid, hydroxyethylstarch (HES), a derivative thereof, or any combinations thereof. Thefirst heterologous moiety (H1) linked to the VWF fragment, the secondheterologous moiety (H2) linked to the FVIII protein, and the thirdheterologous moiety (H3) can be the same or different. In oneembodiment, the first heterologous moiety (H1) is identical to thesecond heterologous moiety (H2), but is different from the thirdheterologous moiety (H3). In another embodiment, the third heterologousmoiety (H3) is fused or linked to a FVIII protein or a VWF fragment ofthe chimeric protein. In some embodiments, the third heterologous moietyis inserted within one or more domains of the FVIII protein or betweentwo domains of the FVIII protein.

In one embodiment, a chimeric protein comprises a first polypeptidechain and a second polypeptide chain, wherein the first chain comprisesa FVIII protein linked to a first heterologous moiety (H1), e.g., afirst Fc region, by an optional linker (e.g., FVIII linker) and thesecond chain comprises a VWF fragment linked to a second heterologousmoiety (H2), e.g., a second Fc region, by an optional linker (e.g., VWFlinker). The FVIII protein can further comprise a third heterologousmoiety (H3), e.g., any half-life extending moiety, e.g., albumin, or aPAS sequence, between FVIII heavy chain and FVIII light chain (i.e.,amino acid residue 1648 of SEQ ID NO: 16), thus being a single chainFVIII protein. Alternatively, the FVIII protein can be a dual chainprotein, i.e., the FVIII heavy chain and the FVIII light chainassociated with each other by a covalent or non-covalent bond (e.g., ametal bond), wherein the heavy chain is further linked to a thirdheterologous moiety (H3), e.g., a non-structural half-life extendingpolypeptide, albumin or a fragment thereof or a PAS sequence. In anotherembodiment, a chimeric protein comprises a first polypeptide chain and asecond polypeptide chain, wherein the first chain comprises a FVIIIprotein linked to a first heterologous moiety (H1), e.g., a first Fcregion, by an optional linker (e.g, FVIII linker) and the second chaincomprises a VWF fragment linked to a third heterologous moiety (H3),e.g., a non-structural half-life extending polypeptide, albumin or a PASsequence, which is linked to a second heterologous moiety (H2), e.g., asecond Fc region, by an optional linker. In some embodiments, the thirdheterologous moiety (H3) (e.g., a half-life extending polypeptide) canbe linked to the C-terminus or N-terminus of the FVIII protein orinserted between two domains of the FVIII protein or between two aminoacids in a domain of the FVIII protein.

In other embodiments, the chimeric protein of the invention furthercomprises a fourth heterologous moiety (sometimes indicated herein by“H4”) and/or a fifth heterologous moiety (sometimes indicated herein by“H5”). The fourth or fifth heterologous moiety can also be a half-lifeextender. The fourth heterologous moiety and/or the fifth heterologousmoiety can be the same or different from the third heterologous moiety.The heterologous moiety can comprise a heterologous polypeptide, anon-polypeptide moiety, or a combination of both. Non-limiting examplesof the fourth or fifth heterologous moiety include an immunoglobulinconstant region or a portion thereof, albumin or a fragment thereof, analbumin binding moiety, a PAS sequence, a HAP sequence, transferrin or afragment thereof, any derivatives or variants thereof, or two or morecombinations thereof. Non-limiting examples of the non-polypeptidemoiety include polyethylene glycol (PEG), polysialic acid, hydroxyethylstarch (HES), a derivative thereof, or any combinations thereof. Thefirst heterologous moiety, the second heterologous moiety, the thirdheterologous moiety, the fourth heterologous moiety, and the fifthheterologous moiety can be the same or different. In some embodiments,the fourth heterologous moiety (e.g., a half-life extending polypeptide)can be linked to the C-terminus or N-terminus of the FVIII protein orinserted between two domains of the FVIII protein or between two aminoacids in a domain of the FVIII protein. In other embodiments, the fifthheterologous moiety (e.g., a half-life extending polypeptide) can alsobe linked to the C-terminus or N-terminus of the FVIII protein orinserted between two domains of the FVIII protein or between two aminoacids in a domain of the FVIII protein.

In certain embodiments, the chimeric protein comprises a FVIII protein,a VWF fragment, a first heterologous moiety, a second heterologousmoiety, a third heterologous moiety, a fourth heterologous moiety, and afifth heterologous moiety, wherein the first heterologous moiety and thesecond heterologous moiety forms a bond (e.g., a covalent bond) betweenthe chain comprising the FVIII protein and the chain comprising the VWFfragment, and the third heterologous moiety, the fourth heterologousmoiety, and the fifth heterologous moiety are half-life extenders, andwherein the bond between the chain comprising the FVIII protein and thechain comprising the VWF fragment is stronger than the non-covalentinteraction between the FVIII and the VWF fragment, thereby preventingbinding of endogenous VWF to the FVIII protein in vivo, in vitro, or exvivo.

In other embodiments, the chimeric protein comprises a FVIII protein, aVWF fragment, a first heterologous moiety, a second heterologous moiety,a third heterologous moiety, a fourth heterologous moiety, a fifthheterologous moiety, and a sixth heterologous moiety (sometimesindicated herein as “H6”), wherein the first heterologous moiety and thesecond heterologous moiety forms a bond between the chain comprising theFVIII protein and the chain comprising the VWF fragment, and the thirdheterologous moiety, the fourth heterologous moiety, the fifthheterologous moiety, and the sixth heterologous moiety are half-lifeextenders, and wherein the bond between the chain comprising the FVIIIprotein and the chain comprising the VWF fragment is stronger than theinteraction between the FVIII and the VWF fragment, thereby preventingbinding of endogenous VWF to the FVIII protein in vivo, in vitro, or exvivo.

In some embodiments, a chimeric protein comprises a formula selectedfrom the group consisting of:

(aa) V-L1-H1-L2-H2, (bb) H2-L2-H1-L1-V, (cc) H1-L1-V-L2-H2, and (dd)H2-L2-V-L1-H1,

wherein V comprises a VWF fragment described herein;

Each of L1 and L2 comprises an optional linker; and

H1 comprises a first heterologous moiety; and

H2 comprises an optional second heterologous moiety. Either or both ofthe first heterologous moiety and the second heterologous moiety can bea half-life extending moiety. In one embodiment, H1 comprises apolypeptide, a non-polypeptide moiety, or both. The polypeptide usefulas H1 can comprise an immunoglobulin constant region or a portionthereof, albumin or a fragment thereof, an albumin binding moiety, a PASsequence, a HAP sequence, any derivatives or variants, or anycombinations thereof. The non-polypeptide moiety can comprisepolyethylene glycol (PEG), polysialic acid, and hydroxyethyl starch(HES), a derivative or variant thereof, or any combinations thereof. Inanother embodiment, H2 comprises a polypeptide, a non-polypeptidemoiety, or both. The polypeptide useful as H2 can comprise animmunoglobulin constant region or a portion thereof, albumin or afragment thereof, an albumin binding moiety, a PAS sequence, a HAPsequence, any derivatives or variants, or any combinations thereof. Thenon-polypeptide moiety can comprise polyethylene glycol (PEG),polysialic acid, hydroxyethyl starch (HES), a derivative or variantthereof, or any combinations thereof. In certain embodiments, the linkerbetween H1 and H2 in formulas (aa) and (bb) is a processable linker. Inother embodiments, the linker between the VWF fragment and H1 informulas (aa) and (bb) is a cleavable linker, e.g., a thrombin cleavablelinker that can be cleaved by thrombin.

The orientation of the polypeptide formulas herein is listed fromN-terminus (left) to C-terminus (right). For example, formula H-L-Vmeans formula NH2-H-L-V-COOH. In one embodiment, the formulas describedherein can comprise additional sequences between the two moieties. Forexample, formula V-L1-H1-L2-H2 can further comprise sequences at theN-terminus of V, between V and L1, between L1 and H1, between H1 or L2,between L2 or H2, or at the C-terminus of H2 unless otherwise specified.In another embodiment, the hyphen (-) indicates a peptide bond or one ormore amino acids.

In specific embodiments, a chimeric protein comprises, consistsessentially of, or consists of one or more formulas selected from thegroup consisting of (a1) V-H, (a2) H-V, (a3) V-L-H, (a4) H-L-V, (a5)V-L1-H1-H2, (a6) H2-H1-L1-V, (a7) V-L1-H1:H2, (a8) H2:H1-L1-V, (a9)V-H1:H2, (b1) H2:H1-V, (b2) V-L1-H1-L2-H2, (b3) H2-L2-H1-L1-V, (b4)H1-V-H2, (b5) H1-L1-V-L2-H2, and (b6) H2-L2-V-L1-H1, wherein V comprisesone or more of the VWF fragments described herein, L, L1, or L2comprises a linker, H or H1 comprises a first heterologous moiety. Inone embodiment, the first heterologous moiety (H1) can be a polypeptide,a non-polypeptide moiety, or both. The heterologous polypeptide moietycan comprises an immunoglobulin constant region or a portion thereof,albumin or a fragment thereof, an albumin binding moiety, a PASsequence, a HAP sequence, or any combinations thereof. Non-limitingexamples of the non-polypeptide moiety useful as H1 include polyethyleneglycol (PEG), polysialic acid, hydroxyethyl starch (HES), a derivativethereof, or any combinations thereof. In another embodiment, H2comprises a second heterologous moiety. The second heterologous moietycan be a polypeptide, a non-polypeptide moiety, or both. Theheterologous polypeptide moiety can comprises an immunoglobulin constantregion or a portion thereof, albumin or a fragment thereof, an albuminbinding moiety, a PAS sequence, a HAP sequence, or any combinationsthereof. Non-limiting examples of the non-polypeptide moiety useful asH1 include polyethylene glycol (PEG), polysialic acid, hydroxyethylstarch (HES), a derivative thereof, or any combinations thereof. Incertain embodiments, the linker between the first heterologous moietyand the second heterologous moiety is a processable linker. In otherembodiments, the linker between the VWF fragment and the firstheterologous moiety or the second heterologous moiety is a cleavablelinker, which comprises one or more cleavage sites, e.g., a thrombincleavable linker.

The chimeric protein of the present invention comprises a formulaselected from the group consisting of (aa), (bb), (cc), (dd), (a1),(a2), (a3), (a4), (a5), (a6), (a7), (a8), (a9), (b1), (b2), (b3), (b4),(b5), and (b6) and a FVIII protein, which is covalently linked to orcovalently associated with the VWF fragment, the first heterologousmoiety (e.g., a first Fc region), or the second heterologous moiety(e.g., a second Fc region) of the formula. In one embodiment, the FVIIIprotein is linked to or associated with the VWF fragment by a covalentor non-covalent bond or by a linker. In another embodiment, the FVIIIprotein can be linked to the first heterologous moiety or the secondheterologous moiety by a covalent or non-covalent bond or by a linker.

In one embodiment, a chimeric protein of the present invention comprisesa VWF fragment described herein covalently linked to or covalentlyassociated with a FVIII protein. For example, the chimeric protein cancomprise a VWF fragment and a FVIII protein, wherein the VWF fragmentand the FVIII protein are bound by a covalent non-peptide bond, apeptide bond, a non-covalent bond, or by a linker, e.g., a cleavablelinker. In a specific embodiment, the VWF fragment and the FVIII proteinare bound to or interact with each other by one or more disulfide bonds.In another specific embodiment, the VWF fragment is bound to orinteracts with the FVIII protein at the A3 domain of FVIII, the C2domain of FVIII, or both the A3 domain and the C2 domain of FVIII by anon-covalent bond. In another embodiment, the VWF fragment bound to orinteracting with the FVIII protein is linked or fused to a firstheterologous moiety. In other embodiments, the FVIII protein bound to orinteracting with the VWF fragment is further linked to a secondheterologous moiety. In some embodiments, the VWF fragment bound to orinteracting with the FVIII protein is further linked to a firstheterologous moiety and the FVIII protein is further linked to a secondheterologous moiety. In certain embodiments, the first polypeptide chaincomprising the VWF fragment and the first heterologous moiety and thesecond polypeptide chain comprising the FVIII protein and the secondheterologous moiety are associated with each other such that theassociation does not allow interaction of the FVIII protein with othermoieties, e.g., endogenous VWF. In one embodiment, the association is acovalent bond, e.g., a disulfide bond.

Each of the VWF fragment or the FVIII protein can be joined or connectedto the first and second heterologous moiety by a linker, e.g., acleavable linker, e.g., a thrombin cleavable linker. The linker betweenthe VWF fragment and the first heterologous moiety can be denoted hereinas a VWF linker. The linker between the FVIII protein and the secondheterologous moiety can be denoted herein as a FVIII linker. Or, both ofthe VWF fragment or the FVIII protein can be joined or connected to thefirst and second heterologous moiety by a linker, e.g., a cleavablelinker, e.g., a thrombin cleavable linker. In certain embodiments, thefirst heterologous moiety linked to the VWF fragment comprises apolypeptide, a non-polypeptide moiety, or both. Non-limiting examples ofthe first heterologous polypeptide moiety includes an immunoglobulinconstant region or a portion thereof, albumin or a fragment thereof, analbumin binding moiety, a PAS sequence, a HAP sequence, transferrin or afragment thereof, or two or more combinations thereof. Non-limitingexamples of the non-polypeptide moiety includes polyethylene glycol(PEG), polysialic acid, hydroxyethyl starch (HES or HAES), a derivativeor variant thereof, or any combinations thereof. In other embodiments,the second heterologous moiety linked to the FVIII protein comprises apolypeptide, a non-polypeptide moiety, or both. Non-limiting examples ofthe second heterologous moiety includes an immunoglobulin constantregion or a portion thereof, albumin or a fragment thereof, an albuminbinding moiety, a PAS sequence, a HAP sequence, transferrin or afragment thereof, or two or more combinations thereof. Non-limitingexamples of the non-polypeptide moiety includes polyethylene glycol(PEG), polysialic acid, hydroxyethyl starch (HES or HAES), a derivativeor variant thereof, or any combinations thereof. In some embodiments,the VWF fragment is attached to FVIII using sortase mediated in vitroprotein ligation. In some embodiments, a sortase recognition motif isused.

In one embodiment, the first heterologous moiety is an immunoglobulinconstant region or a portion thereof. In a particular embodiment, thefirst heterologous moiety is a first Fc region. In some embodiments, thesecond heterologous moiety is an immunoglobulin constant region or aportion thereof. In a specific embodiment, the second heterologousmoiety is a second Fc region. In a particular embodiment, the chimericprotein comprises a VWF fragment described herein and a FVIII protein,wherein the VWF fragment is linked to an immunoglobulin constant regionor a portion thereof, which is an Fc region. In another embodiment, thechimeric protein comprises a VWF fragment described herein and a FVIIIprotein, wherein the FVIII protein is linked to an immunoglobulinconstant region or a portion thereof, which is an Fc region. In otherembodiments, a chimeric protein comprises a VWF fragment describedherein and a FVIII protein, wherein the VWF fragment is linked to afirst immunoglobulin constant region, which is a first Fc region, andthe FVIII protein is linked to a second immunoglobulin constant region,which is a second Fc region, and wherein the VWF fragment and the FVIIIprotein is bound to or interact with each other by a non-covalent bondor the first Fc region or the second Fc region are associated with eachother by a covalent bond. In still other embodiments, the VWF fragmentlinked to the first heterologous moiety is further linked to the secondheterologous moiety, e.g., a second Fc region, by a linker, e.g., aprocessable linker. In one aspect, the VWF fragment is linked to thefirst heterologous moiety by a linker, e.g., VWF linker, e.g., acleavable linker. In another aspect, the FVIII protein is linked to thesecond heterologous moiety by a linker, e.g., FVIII linker, e.g., acleavable linker. Non-limiting examples of the heterologous moieties aredisclosed elsewhere herein, e.g., immunoglobulin constant region or aportion thereof at paragraphs [0165]-[0193], albumin, fragment orvariant thereof at paragraphs [0194]-[0198], HAP sequences at paragraph[0293], transferrin, fragments, or variants thereof at paragraphs[0204]-[0205], polymer, e.g., polyethylene glycol, at paragraphs[0206]-[0213], HES at paragraphs [0214]-[0219], or PSA at paragraph[0220]- and PAS sequences at paragraphs [0199]-[0202].

In some embodiments, a chimeric protein of the present inventioncomprises, consists essentially of, or consists of a formula selectedfrom the group consisting of:

(a) V-L1-H1-L3- C-L2-H2, (b) H2-L2-C-L3- H1-L1-V, (c) C-L2-H2- L3-V-L1-H1, (d) H1-L1-V-L3-H2-L2-C, (e) H1-L1-V-L3-C-L2-H2, (g) H2-L2-C-L3-V-L1-H1, (g) V-L1-H1-L3- H2-L2-C, (g) C-L2-H2- L3- H1-L1-V, (i)H2-L3-H1-L1-V-L2-C, (j) C-L2-V-L1-H1-L3-H2, (k) V-L2-C-L1-H1-L3-H2, and(l) H2-L3-H1-L1-C-L2-V,

wherein V is a VWF fragment described herein;

each of L1 or L2 is an optional linker, e.g., a cleavable linker, e.g.,a thrombin cleavable linker;

L3 is an optional linker, e.g., a processable linker

each of H1 and H2 is an optional heterologous moiety;

C is a FVIII protein; and

(-) is a peptide bond or one or more amino acids.

In other aspects, a chimeric protein of the invention comprises aformula selected from the group consisting of:

(m) V-L1-H1:H2-L2-C, (n) V-L1-H1:C-L2-H2; (o) H1-L1-V:H2-L2-C; (p)H1-L1-V:C-L2-H2; (q) V:C-L1-H1:H2; (r) V:H1-L1-C:H2; (s) H2:H1-L1-C:V,(t) C:V-L1-H1:H2, and (u) C:H1-L1-V:H2.

wherein V is a VWF fragment described herein;

each of L1 or L2, is an optional linker, e.g., a thrombin cleavablelinker;

each of H1 or H2 is an optional heterologous moiety;

(-) is a peptide bond or one or more amino acids; and

C is a FVIII protein; and (:) is a chemical or physical associationbetween H1 and H2.

In one embodiment, one or more of the heterologous moieties are ahalf-life extender. Half-life extenders are known in the art, andnon-limiting examples of such half-life extenders include animmunoglobulin constant region or a portion thereof, albumin or fragmentthereof, an albumin binding moiety, a PAS sequence, a HAP sequence,transferrin or a fragment thereof, a derivative or variant thereof, ortwo or more combinations thereof. The non-polypeptide moiety cancomprise polyethylene glycol (PEG), polysialic acid, hydroxyethyl starch(HES), a derivative thereof, or any combinations thereof.

In one embodiment, (:) in formulas (m) to (u) represents a chemicalassociation, e.g., at least one non-peptide bond. In certainembodiments, the chemical association, i.e., (:) is a covalent bond. Inother embodiments, the chemical association, i.e., (:) is a non-covalentinteraction, e.g., an ionic interaction, a hydrophobic interaction, ahydrophilic interaction, a Van der Waals interaction, a hydrogen bond.In other embodiments, (:) is a non-peptide covalent bond. In still otherembodiments, (:) is a peptide bond. In yet other embodiments, (:) informulas (m) to (u) represents a physical association between twosequences, wherein a portion of a first sequence is in close proximityto a second sequence such that the first sequence shields or blocks aportion of the second sequence from interacting with another moiety, andfurther that this physical association is maintained without allowingthe second sequence to interact with other moieties.

Formulas (a)-(u) are included herein merely as non-limiting examples ofconstructs of the present invention. The orientation of the polypeptideformulas is shown from N-terminus (left) to C-terminus (right). Forexample, formula V-L1-H1-L3-C-L2-H2 means formulaNH2-V-L1-H1-L3-C-L2-H2-COOH. In addition, (:) can be an association orinteraction between two polypeptide chains by a covalent bond or anon-covalent bond between any part of the first chain and any part ofthe second chain unless otherwise noted. For example, formula V-H1:H2-Chas two polypeptide chains, the first chain being V-H1 and the secondchain being C-H2, wherein V in the first chain interacts or associateswith C in the second chain and/or H1 in the first chain interacts orassociates with H2 in the second chain. In some embodiments, (:) means acovalent, non-peptide bond or non-covalent bond.

In certain embodiments, a chimeric protein comprises, consistsessentially of, or consists of a formula selected from the groupconsisting of:

 (1) V:C,  (2) H—V:C or C:V—H,  (3) V:C—H or H—C:V,  (4) V—H1:H2—C orH1—V:C—H2,  (5) V:C—H1:H2 or H2:H1—C:V,  (6) H2:H1—V:C or C:V—H1:H2, (7) H—L—V:C or C:V—L—H,  (8) V:C—L—H or H—L—C:V,  (9) V—C or C—V, (10)H—V—C or C—V—H, (11) V—H—C or C—H—V, (12) V—C—H or H—C—V, (13) V—H1—C—H2or H2—C—H1—V, (14) H1—V—C—H2 or H2—C—V—H1, (15) H1—V—H2—C or C—H2—V—H1,(16) V—H1—H2—C or C—H2—H1—V, (17) V—L—C or C—L—V, (18) H—L—V—C orC—V—L—H, (19) H—V—L—C or C—L—V—H, (20) V—L—H—C or C—H—L—V, (21) V—H—L—Cor C—L—H—V, (22) V—L—C—H or H—C—L—V, (23) V—C—L—H or H—L—C—V, (24)H—L1—V—L2—C or C—L2—V—L1—H, (25) V—L—H1:H2—C or C—H2:H1—L—V, (26)V—H1:H2—L—C or C—L—H2:H1—V, (27) V:C—H1—H2 or H2—H1—C:V, (28) H2—H1—V:Cor C:V—H1—H2, (29) V:C—L—H1:H2 or H2:H1—L—C:V, (30) H2:H1—L—V:C orC:V—L—H1:H2, (31) V—L1—H1:H2—L2—C or L—L2—H2:H1—L1—V, (32) V:C—L—H1—H2or H2—H1—L—C:V, (33) V:C—H1—L—H2 or H2—L—H1—C:V, (34) V:C—L1—H1—L2—H2 orH2—L2—H1—L1—C:V, (35) H2—H1—V:C or C:V—H1—H2, (36) H2—H1—L—V:C orC:V—L—H1—H2, (37) H2—L—H1—V:C or C:V—H1—L—H2, (38) H2—L2—H1—L1—V:C orC:V—L1—H1—L2—H2, (39) V—L1—H—L2—C or C—L2—H—L1—V, (40) V—L1—C—L2—H orH—L2—C—L1—V, (41) V—L—H1—C—H2 or H2—C—H1—L—V, (42) V—H1—C—L—H2 orH2—L—C—H1—V, (43) V—H1—L—C—H2 or H2—C—L—H1—V, (44) H1—L—V—C—H2 orH2—C—V—L—H1, (45) H1—V—L—C—H2 or H2—C—L—V—H1, (46) H1—V—C—L—H orH—L—C—V—H1, (47) H1—L—V—H2—C or C—H2—V—L—H1, (48) H1—V—L—H2—C orC—H2—L—V—H1, (49) H1—V—H2—L—C or C—L—H2—V—H1, (50) V—L—H1—H2—C orC—H2—H1—L—V, (51) V—H1—L—H2—C or C—H2—L—H1—V, (52) V—H1—H2—L—C orC—L—H2—H1—V, (53) V—L1—H1—L2—C—H2 or H2—C—L2—H1—L1—V, (54)V—L1—H1—C—L2—H2 or H2—L2—C—H1—L1—V, (55) V—L1—H1—L2—C—L3—H2 orH2—L3—C—L2—H1—L1—V, (56) V—H1—L1—C—L2—H2 or H2—L2—C—L1—H1—V, (57)H1—L1—V—L2—C—H2 or H2—C—L2—V—L1—H1, (58) H1—L1—V—C—L2—H2 orH2—L2—C—V—L1—H1, (59) H1—L1—V—L2—C—L3—H2 or H2—L3—C—L2—V—L1—H1, (60)H1—V—L1—C—L2—H2 or H2—L2—C—L1—V—H1, (61) H1—L1—V—L2—H2—C orC—H2—L2—V—L1—H1, (62) H1—L1—V—H2—L2—C or C—L2—H2—V—L1—H1, (63)H1—L1—V—L2—H2—L3—C or C—L3—H2—L2—V—L1—H1, (64) H1—V—L1—H2—L2—C orC—L2—H2—L1—V—H1, (65) V—L1—H1—L2—H2—C or C—H2—L2—H1—L1—V, (66)V—L1—H1—H2—L2—C or C—L2—H2—H1—L1—V, (67) V—L1—H1—L2—H2—L3—C orC—L3—H2—L2—H1—L1—V, and (68) V—H1—L1—H2—L2—C or C—L2—H2—L1—H1—V,V is a VWF fragment described herein;C is a FVIII protein;H or H1 is a heterologous moiety or a first heterologous moiety;H2 is a second heterologous moiety; the first and second heterologousmoieties can be the same or different;Each of L, L1 or L2 is an optional linker;(-) is a peptide bond or one or more amino acids; and

(:) is a chemical or physical association. The linkers can each be thesame or different and each can be a cleavable linker, comprising one ormore enzymatic cleavage site. The heterologous moieties can be ahalf-life extension technology that is known in the art, a polypeptide,a non-polypeptide moiety, or both. A polypeptide moiety can comprise animmunoglobulin constant region or a portion thereof, albumin or afragment thereof, an albumin binding moiety, a PAS sequence, a HAPsequence, any derivatives or variants thereof, or any combinationsthereof (e.g., an Fc region). A non-polypeptide moiety can comprisepolyethylene glycol (PEG), polysialic acid, hydroxyethyl starch (HES), aderivative or variant thereof, or any combinations thereof. Each of theH, H1, or H2 can be individually selected based on the characteristicsand can be all the same, or each one different. Non-limiting examples ofthe heterologous moieties are disclosed elsewhere herein, e.g.,immunoglobulin constant region or a portion thereof at paragraphs[0126]-[0153], albumin or fragment or variant thereof at paragraphs[0154]-[0157], polymer, e.g., polyethylene glycol, at paragraphs[0166]-[0173], and PAS sequences at paragraphs [0159]-[0162]. Formulas(1)-(68) are included herein merely as non-limiting examples ofconstructs of the present invention.

In one embodiment, (:) represents a chemical association, e.g., at leastone non-peptide bond. In certain embodiments, the chemical association,i.e., (:) is a covalent bond. In other embodiments, the chemicalassociation, i.e., (:) is a non-covalent interaction, e.g., an ionicinteraction, a hydrophobic interaction, a hydrophilic interaction, a Vander Waals interaction, a hydrogen bond. In other embodiments, (:) is anon-peptide covalent bond. In still other embodiments, (:) is a peptidebond. In yet other embodiments, (:) represents a physical associationbetween two sequences, wherein a portion of a first sequence is in closeproximity to a second sequence such that the first sequence shields orblocks a portion of the second sequence from interacting with anothermoiety, and further that this physical association is maintained withoutallowing the second sequence to interact with other moieties.

In one embodiment, the first heterologous moiety (H or H1) linked to theVWF fragment in the chimeric protein is a first Fc region. In anotherembodiment, the second heterologous moiety (or H2) linked to the FVIIIprotein in the chimeric protein is a second Fc region.

In certain embodiments, a chimeric protein of the invention comprisestwo polypeptide chains, a first chain comprising, consisting essentiallyof, or consisting of an amino acid sequence encoding FVIII (e.g., singlechain FVIII) and a first heterologous moiety (e.g., a first Fc region)and a second chain comprising, consisting essentially of, or consistingof an amino acid sequence encoding a VWF fragment comprising D′ domainand D3 domain, a second heterologous moiety (e.g., a second Fc region),and a linker between the VWF fragment and the second Fc domain (e.g.,VWF linker). The linker between the VWF fragment and the second Fcdomain can be a thrombin cleavable linker. In some embodiments, thesingle chain FVIII protein comprises a third heterologous moiety, e.g.,a half-life extender, which is linked to the N-terminus, C-terminus, orone or more sites within the FVIII sequence.

In other embodiments, a chimeric protein of the invention comprisesthree polypeptide chains, wherein a first chain comprises, consistsessentially of, or consists of a heavy chain of FVIII, a second chaincomprises, consists essentially of, or consists of a light chain ofFVIII fused to a first heterologous moiety (e.g., a first Fc region),and a third polypeptide chain comprises, consists essentially of, orconsists of a VWF fragment comprising the D′ domain and the D3 domain, asecond heterologous moiety (e.g, a second Fc region), and a linker. Thelinker between the VWF fragment and the second heterologous moiety canbe a thrombin cleavable linker. In some embodiments, the heavy chainFVIII is linked to a third heterologous moiety, e.g., a half-lifeextender, which can be linked to the N-terminus, C-terminus, or one ormore sites within the FVIII sequence.

In yet other embodiments, a chimeric protein of the invention comprisestwo polypeptide chains, a first chain comprising, consisting essentiallyof, or consisting of a heavy chain of FVIII and a second chaincomprising, consisting essentially of, or consisting of a light chain ofFVIII, a first heterologous moiety (e.g., a first Fc region), a firstlinker (e.g., a protease cleavage site comprising one or moreintracellular processing sites), a VWF fragment, a second linker (e.g.,a thrombin cleavable linker), and a second heterologous moiety (e.g., asecond Fc region), wherein the light chain of FVIII is linked to thefirst heterologous moiety (e.g., the first Fc region), which is furtherlinked to the VWF fragment by the first linker (e.g. a processablelinker having a protease cleavage site comprising one or moreintracellular processing sites), and wherein the VWF fragment is linkedto the second Fc region by the second linker (e.g., a thrombin cleavablelinker). In certain embodiments, the first linker and the second linkerare identical or different.

In certain embodiments, a chimeric protein of the invention comprisesone polypeptide chain, which comprises a single chain FVIII protein, afirst heterologous moiety (e.g., a first Fc region), a first linker(e.g., a thrombin cleavable linker), a VWF fragment, a second linker(e.g., a thrombin cleavable linker), and a second heterologous moiety(e.g., a second Fc region), wherein the single chain FVIII protein islinked to the first heterologous moiety, which is also linked to the VWFfragment by the first linker, and the VWF fragment is linked to thesecond Fc region by the second linker. In one embodiment, the firstlinker is a cleavable linker comprising a first cleavable site and asecond cleavable site. In another embodiment, the second linker is acleavable linker comprising one or two cleavable sites. In a specificembodiment, the second linker is a thrombin cleavable linker. The linkeruseful in the invention can be any length, e.g., at least 10, 50, 100,200, 300, 400, 500, 600, or 700 amino acids. For example, the linker canbe 20 amino acids, 35 amino acids, 42 amino acids, 73 amino acids, or 98amino acids.

In certain embodiments, the VWF fragment is directly linked to the FVIIIprotein by a peptide bond or a linker. As one way of linking the VWFfragment and the FVIII protein directly or through a linker, anenzymatic ligation (e.g., sortase) can be employed. For example, sortaserefers to a group of prokaryotic enzymes that modify surface proteins byrecognizing and cleaving a carboxyl-terminal sorting signal. For mostsubstrates of sortase enzymes, the recognition signal consists of themotif LPXTG (Leu-Pro-any-Thr-Gly (SEQ ID NO: 106), then a highlyhydrophobic transmembrane sequence, then a cluster of basic residuessuch as arginine. Cleavage occurs between the Thr and Gly, withtransient attachment through the Thr residue to the active site Cysresidue of a ligation partner, followed by transpeptidation thatattaches the protein covalently to the cell wall. In some embodiments,the ligation partner contains Gly(n).

In one embodiment, a VWF fragment linked to a sortase recognition motifby an optional linker can be fused to a FVIII protein linked to Gly(n)by a sortase, wherein n can be any integer. A ligation constructcomprises the VWF fragment (N-terminal portion of the construct) and theFVIII protein (C-terminal portion of the construct), wherein the sortaserecognition motif is inserted in between. An exemplary construct isshown in FIG. 24(A). Another ligation construct comprises the VWFfragment (N-terminal portion of the construct, the linker, the sortaserecognition motif, and the FVIII protein (C-terminal portion of theconstruct) (e.g., FIG. 24(C)). In another embodiment, a FVIII proteinlinked to a sortase recognition motif by an optional linker can be fusedto a VWF fragment linked to Gly(n) by a sortase, wherein n is anyinteger. A resulting ligation construct comprises the FVIII protein(N-terminal portion of the construct) and the VWF fragment (C-terminalportion of the construct), wherein the sortase recognition motif isinserted in between. An exemplary construct is shown in FIG. 24(B).Another resulting ligation construct comprises the FVIII protein(N-terminal portion of the construct), the linker, the sortaserecognition motif, and the VWF fragment (C-terminal portion of theconstruct) (e.g., FIG. 24(D)). In other embodiments, a VWF fragmentlinked to a sortase recognition motif by a first optional linker can befused to a heterologous moiety, e.g., an immunoglobulin constant regionor a portion thereof, e.g., an Fc region, linked to a thrombin cleavagesite by a second optional linker. A resulting construct can comprise theVWF fragment (N-terminal portion), the first linker, the sortaserecognition motif, the protease cleavage site, the second optionallinker, and the heterologous moiety (e.g., FIG. 24(E)). In certainembodiments, this resulting construct is a part of a chimeric proteincomprising the FVIII protein and a second heterologous moiety, e.g., animmunoglobulin constant region or a portion thereof, e.g., a second Fcregion. In one example, In another example, a chimeric comprises threepolypeptide chains, the first chain comprising a VWF fragment, the firstlinker, the sortase recognition motif, the protease cleavage site, thesecond optional linker, the first heterologous moiety, the second chaincomprising the light chain of the FVIII protein and the secondheterologous moiety, and the third chain comprising the heavy chain ofthe FVIII protein.

In still other embodiments, the chimeric protein of the inventioncomprising a VWF fragment and a FVIII protein, wherein the VWF fragmentand the FVIII protein are covalently associated with each other orcovalently linked to each other has less immunogenicity than a FVIIIprotein without the VWF fragment. The reduced immunogenicity includes,but is not limited to, less humoral immune response, e.g., lessneutralizing antibody titer, or less cell-mediated immune responseagainst FVIII, e.g., production of various cytokines.

In yet other embodiments, as a result of the invention the half-life ofthe FVIII protein (or a chimeric protein) is extended compared to aFVIII protein without the VWF fragment or wildtype FVIII. The half-lifeof the FVIII protein is at least about 1.5 times, at least about 2times, at least about 2.5 times, at least about 3 times, at least about4 times, at least about 5 times, at least about 6 times, at least about7 times, at least about 8 times, at least about 9 times, at least about10 times, at least about 11 times, or at least about 12 times longerthan the half-life of a FVIII protein without the VWF fragment. In oneembodiment, the half-life of FVIII is about 1.5-fold to about 20-fold,about 1.5 fold to about 15 fold, or about 1.5 fold to about 10 foldlonger than the half-life of wild-type FVIII. In another embodiment, thehalf-life of the FVIII is extended about 2-fold to about 10-fold, about2-fold to about 9-fold, about 2-fold to about 8-fold, about 2-fold toabout 7-fold, about 2-fold to about 6-fold, about 2-fold to about5-fold, about 2-fold to about 4-fold, about 2-fold to about 3-fold,about 2.5-fold to about 10-fold, about 2.5-fold to about 9-fold, about2.5-fold to about 8-fold, about 2.5-fold to about 7-fold, about 2.5-foldto about 6-fold, about 2.5-fold to about 5-fold, about 2.5-fold to about4-fold, about 2.5-fold to about 3-fold, about 3-fold to about 10-fold,about 3-fold to about 9-fold, about 3-fold to about 8-fold, about 3-foldto about 7-fold, about 3-fold to about 6-fold, about 3-fold to about5-fold, about 3-fold to about 4-fold, about 4-fold to about 6 fold,about 5-fold to about 7-fold, or about 6-fold to about 8 fold ascompared to wild-type FVIII or a FVIII protein without the VWF fragment.In other embodiments, the half-life of FVIII is at least about 17 hours,at least about 18 hours, at least about 19 hours, at least about 20hours, at least about 21 hours, at least about 22 hours, at least about23 hours, at least about 24 hours, at least about 25 hours, at leastabout 26 hours, at least about 27 hours, at least about 28 hours, atleast about 29 hours, at least about 30 hours, at least about 31 hours,at least about 32 hours, at least about 33 hours, at least about 34hours, at least about 35 hours, at least about 36 hours, at least about48 hours, at least about 60 hours, at least about 72 hours, at leastabout 84 hours, at least about 96 hours, or at least about 108 hours. Instill other embodiments, the half-life of FVIII is about 15 hours toabout two weeks, about 16 hours to about one week, about 17 hours toabout one week, about 18 hours to about one week, about 19 hours toabout one week, about 20 hours to about one week, about 21 hours toabout one week, about 22 hours to about one week, about 23 hours toabout one week, about 24 hours to about one week, about 36 hours toabout one week, about 48 hours to about one week, about 60 hours toabout one week, about 24 hours to about six days, about 24 hours toabout five days, about 24 hours to about four days, about 24 hours toabout three days, or about 24 hours to about two days.

In some embodiments, the average half-life of the FVIII protein persubject is about 15 hours, about 16 hours, about 17 hours, about 18hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours,about 23 hours, about 24 hours (1 day), about 25 hours, about 26 hours,about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours,about 36 hours, about 40 hours, about 44 hours, about 48 hours (2 days),about 54 hours, about 60 hours, about 72 hours (3 days), about 84 hours,about 96 hours (4 days), about 108 hours, about 120 hours (5 days),about six days, about seven days (one week), about eight days, aboutnine days, about 10 days, about 11 days, about 12 days, about 13 days,or about 14 days.

In certain embodiments, the half-life of the FVIII protein covalentlylinked to the VWF fragment is extendable in FVIII/VWF double knockout(“DKO”) mice compared to a polypeptide consisting of FVIII or a FVIIImonomer-dimer hybrid.

A) Von Willebrand Factor (VWF) Fragments

VWF (also known as F8VWF) is a large multimeric glycoprotein present inblood plasma and produced constitutively in endothelium (in theWeibel-Palade bodies), megakaryocytes (α-granules of platelets), andsubendothelian connective tissue. The basic VWF monomer is a 2813 aminoacid protein. Every monomer contains a number of specific domains with aspecific function, the D′ and D3 domains (which together bind to FactorVIII), the A1 domain (which binds to platelet GPIb-receptor, heparin,and/or possibly collagen), the A3 domain (which binds to collagen), theC1 domain (in which the RGD domain binds to platelet integrin αIIbβ3when this is activated), and the “cysteine knot” domain at theC-terminal end of the protein (which VWF shares with platelet-derivedgrowth factor (PDGF), transforming growth factor-β (TGFβ) and β-humanchorionic gonadotropin (βHCG)).

The 2813 monomer amino acid sequence for human VWF is reported asAccession Number _NP_000543.2_ in Genbank. The nucleotide sequenceencoding the human VWF is reported as Accession Number _NM _000552.3 inGenbank. The nucleotide sequence of human VWF is designated as SEQ IDNO: 1. SEQ ID NO: 2 is the amino acid sequence encoded by SEQ ID NO: 1.Each domain of VWF is listed in Table 1.

TABLE 1 VWF domains Amino acid Sequence VWF Signal Peptide    1MIPARFAGVL LALALILPGT LC                 22 (Amino acids 1 to 22 ofSEQ ID NO: 2) VWF D1D2 region   23                        AEGTRGRS STARCSLFGS (Amino acids 23 to 763DFVNTFDGSM of SEQ ID NO: 2)   51YSFAGYCSYL LAGGCQKRSF SIIGDFQNGK RVSLSVYLGE FFDIHLFVNG  101TVTQGDQRVS MPYASKGLYL ETEAGYYKLS GEAYGFVARI DGSGNFQVLL  151SDRYFNKTCG LCGNFNIFAE DDFMTQEGTL TSDPYDFANS WALSSGEQWC  201ERASPPSSSC NISSGEMQKG LWEQCQLLKS TSVFARCHPL VDPEPFVALC  251EKTLCECAGG LECACPALLE YARTCAQEGM VLYGWTDHSA CSPVCPAGME  301YRQCVSPCAR TCQSLHINEM CQERCVDGCS CPEGQLLDEG LCVESTECPC  351VHSGKRYPPG TSLSRDCNTC ICRNSQWICS NEECPGECLV TGQSHFKSFD  401NRYFTFSGIC QYLLARDCQD HSFSIVIETV QCADDRDAVC TRSVTVRLPG  451LHNSLVKLKH GAGVAMDGQD IQLPLLKGDL RIQHTVTASV RLSYGEDLQM  501DWDGRGRLLV KLSPVYAGKT CGLCGNYNGN QGDDFLTPSG LAEPRVEDFG  551NAWKLHGDCQ DLQKQHSDPC ALNPRMTRFS EEACAVLTSP TFEACHRAVS  601PLPYLRNCRY DVCSCSDGRE CLCGALASYA AACAGRGVRV AWREPGRCEL  651NCPKGQVYLQ CGTPCNLTCR SLSYPDEECN EACLEGCFCP PGLYMDERGD  701CVPKAQCPCY YDGEIFQPED IFSDHHTMCY CEDGFMHCTM SGVPGSLLPD  751AVLSSPLSHR SKR                          763 VWF D′ Domain(Amino acids 764 to 866 of SEQ ID NO: 2)  764

 801

 851

VWF D3 Domain (Amino acids 867 to 1240 of SEQ ID NO: 2)  867

 901

 951

1001

1051

1101

1151

1201

1240 VWF A1 Domain 1241 GGLVVPPTDA (Amino acids 1241 to 1251PVSPTTLYVE DISEPPLHDF YCSRLLDLVF LLDGSSRLSE 1479 of SEQ ID NO: 2)AEFEVLKAFV 1301 VDMMERLRIS QKWVRVAVVE YHDGSHAYIG LKDRKRPSEL RRIASQVKYA1351 GSQVASTSEV LKYTLFQIFS KIDRPEASRI ALLLMASQEP QRMSRNFVRY 1401VQGLKKKKVI VIPVGIGPHA NLKQIRLIEK QAPENKAFVL SSVDELEQQR 1451DEIVSYLCDL APEAPPPTLP PDMAQVTVG        1479 1480                       P GLLGVSTLGP KRNSMVLDVA 1501FVLEGSDKIG EADFNRSKEF MEEVIQRMDV GQDSIHVTVL QYSYMVTVEY 1551PFSEAQSKGD ILQRVREIRY QGGNRTNTGL ALRYLSDHSFLVSQGDREQA                             1600 1601PNLVYMVTGN PASDEIKRLP GDIQVVPIGV GPNANVQELE RIGWPNAPIL 1651IQDFETLPRE APDLVLQRCC SGEGLQIPTL SPAPDCSQPL DVILLLDGSS 1701SFPASYFDEM KSFAKAFISK ANIGPRLTQV SVLQYGSITT IDVPWNVVPE 1751KAHLLSLVDV MQREGGPSQI GDALGFAVRY LTSEMHGARP GASKAVVILV 1801TDVSVDSVDA AADAARSNRV TVFPIGIGDR YDAAQLRILA GPAGDSNVVK 1851LQRIEDLPTM VTLGNSFLHK LCSGFVRICM DEDGNEKRPG DVWTLPDQCH 1901TVTCQPDGQT LLKSHRVNCD RGLRPSCPNS QSPVKVEETC GCRWTCPCVC 1951TGSSTRHIVT FDGQNFKLTG SCSYVLFQNK EQDLEVILHN GACSPGARQG 2001CMKSIEVKHS ALSVEXHSDM EVTVNGRLVS VPYVGGNMEV NVYGAIMHEV 2051RFNHLGHIFT FTPQNNEFQL QLSPKTFASK TYGLCGICDE NGANDFMLRD 2101GTVTTDWKTL VQEWTVQRPG QTCQPILEEQ CLVPDSSHCQ VLLLPLFAEC 2151HKVLAPATFY AICQQDSCHQ EQVCEVIASY AHLCRTNGVC VDWRTPDFCA 2201MSCPPSLVYN HCEHGCPRHC DGNVSSCGDH PSEGCFCPPD KVMLEGSCVP 2251EEACTQCIGE DGVQHQFLEA WVPDHQPCQI CTCLSGRKVN CTTQPCPTAK 2301APTCGLCEVA RLRQNADQCC PEYECVCDPV SCDLPPVPHC ERGLQPTLTN 2351PGECRPNFTC ACRKEECKRV SPPSCPPHRL PTLRKTQCCD EYECACNCVN 2401STVSCPLGYL ASTATNDCGC TTTTCLPDKV CVHRSTIYPV GQFWEEGCDV 2451CTCTDMEDAV MGLRVAQCSQ KPCEDSCRSG FTYVLHEGEC CGRCLPSACE 2501VVTGSPRGDS QSSWKSVGSQ WASPENPCLI NECVRVKEEV FIQQRNVSCP 2551QLEVPVCPSG FQLSCKTSAC CPSCRCERME ACMLNGTVIG PGKTVMIDVC 2601TTCRCMVQVG VISGFKLECR KTTCNPCPLG YKEENNTGEC CGRCLPTACT 2651IQLRGGQIMT LKRDETLQDG CDTHFCKVNE RGEYFWEKRV TGCPPFDEHK 2701CLAEGGKIMK IPGTCCDTCE EPECNDITAR LQYVKVGSCK SEVEVDIHYC 2751QGKCASKAMY SIDINDVQDQ CSCCSPTRTE PMQVALHCTN GSVVYHEVLN 2801AMECKCSPRK CSK Nucleotide Sequence Full-length VWFATGATTCCTG CCAGATTTGC CGGGGTGCTG CTTGCTCTGG CCCTCATTTT (SEQ ID NO: 1)GCCAGGGACC CTTTGTGCAG AAGGAACTCG CGGCAGGTCA TCCACGGCCCTACTAAGGAC GGTCTAAACG GCCCCACGAC GAACGAGACC GGGAGTAAAACGGTCCCTGG GAAACACGTC TTCCTTGAGC GCCGTCCAGT AGGTGCCGGGGATGCAGCCT TTTCGGAAGT GACTTCGTCA ACACCTTTGA TGGGAGCATGTACAGCTTTG CGGGATACTG CAGTTACCTC CTGGCAGGGG GCTGCCAGAACTACGTCGGA AAAGCCTTCA CTGAAGCAGT TGTGGAAACT ACCCTCGTACATGTCGAAAC GCCCTATGAC GTCAATGGAG GACCGTCCCC CGACGGTCTTACGCTCCTTC TCGATTATTG GGGACTTCCA GAATGGCAAG AGAGTGAGCCTCTCCGTGTA TCTTGGGGAA TTTTTTGACA TCCATTTGTT TGTCAATGGTTGCGAGGAAG AGCTAATAAC CCCTGAAGGT CTTACCGTTC TCTCACTCGGAGAGGCACAT AGAACCCCTT AAAAAACTGT AGGTAAACAA ACAGTTACCAACCGTGACAC AGGGGGACCA AAGAGTCTCC ATGCCCTATG CCTCCAAAGGGCTGTATCTA GAAACTGAGG CTGGGTACTA CAAGCTGTCC GGTGAGGCCTTGGCACTGTG TCCCCCTGGT TTCTCAGAGG TACGGGATAC GGAGGTTTCCCGACATAGAT CTTTGACTCC GACCCATGAT GTTCGACAGG CCACTCCGGAATGGCTTTGT GGCCAGGATC GATGGCAGCG GCAACTTTCA AGTCCTGCTGTCAGACAGAT ACTTCAACAA GACCTGCGGG CTGTGTGGCA ACTTTAACATTACCGAAACA CCGGTCCTAG CTACCGTCGC CGTTGAAAGT TCAGGACGACAGTCTGTCTA TGAAGTTGTT CTGGACGCCC GACACACCGT TGAAATTGTACTTTGCTGAA GATGACTTTA TGACCCAAGA AGGGACCTTG ACCTCGGACCCTTATGACTT TGCCAACTCA TGGGCTCTGA GCAGTGGAGA ACAGTGGTGTGAAACGACTT CTACTGAAAT ACTGGGTTCT TCCCTGGAAC TGGAGCCTGGGAATACTGAA ACGGTTGAGT ACCCGAGACT CGTCACCTCT TGTCACCACAGAACGGGCAT CTCCTCCCAG CAGCTCATGC AACATCTCCT CTGGGGAAATGCAGAAGGGC CTGTGGGAGC AGTGCCAGCT TCTGAAGAGC ACCTCGGTGTCTTGCCCGTA GAGGAGGGTC GTCGAGTACG TTGTAGAGGA GACCCCTTTACGTCTTCCCG GACACCCTCG TCACGGTCGA AGACTTCTCG TGGAGCCACATTGCCCGCTG CCACCCTCTG GTGGACCCCG AGCCTTTTGT GGCCCTGTGTGAGAAGACTT TGTGTGAGTG TGCTGGGGGG CTGGAGTGCG CCTGCCCTGCAACGGGCGAC GGTGGGAGAC CACCTGGGGC TCGGAAAACA CCGGGACACACTCTTCTGAA ACACACTCAC ACGACCCCCC GACCTCACGC GGACGGGACGCCTCCTGGAG TACGCCCGGA CCTGTGCCCA GGAGGGAATG GTGCTGTACGGCTGGACCGA CCACAGCGCG TGCAGCCCAG TGTGCCCTGC TGGTATGGAGGGAGGACCTC ATGCGGGCCT GGACACGGGT CCTCCCTTAC CACGACATGCCGACCTGGCT GGTGTCGCGC ACGTCGGGTC ACACGGGACG ACCATACCTCTATAGGCAGT GTGTGTCCCC TTGCGCCAGG ACCTGCCAGA GCCTGCACATCAATGAAATG TGTCAGGAGC GATGCGTGGA TGGCTGCAGC TGCCCTGAGGATATCCGTCA CACACAGGGG AACGCGGTCC TGGACGGTCT CGGACGTGTAGTTACTTTAC ACAGTCCTCG CTACGCACCT ACCGACGTCG ACGGGACTCCGACAGCTCCT GGATGAAGGC CTCTGCGTGG AGAGCACCGA GTGTCCCTGCGTGCATTCCG GAAAGCGCTA CCCTCCCGGC ACCTCCCTCT CTCGAGACTGCTGTCGAGGA CCTACTTCCG GAGACGCACC TCTCGTGGCT CACAGGGACGCACGTAAGGC CTTTCGCGAT GGGAGGGCCG TGGAGGGAGA GAGCTCTGACCAACACCTGC ATTTGCCGAA ACAGCCAGTG GATCTGCAGC AATGAAGAATGTCCAGGGGA GTGCCTTGTC ACTGGTCAAT CCCACTTCAA GAGCTTTGACGTTGTGGACG TAAACGGCTT TGTCGGTCAC CTAGACGTCG TTACTTCTTACAGGTCCCCT CACGGAACAG TGACCAGTTA GGGTGAAGTT CTCGAAACTGAACAGATACT TCACCTTCAG TGGGATCTGC CAGTACCTGC TGGCCCGGGATTGCCAGGAC CACTCCTTCT CCATTGTCAT TGAGACTGTC CAGTGTGCTGTTGTCTATGA AGTGGAAGTC ACCCTAGACG GTCATGGACG ACCGGGCCCTAACGGTCCTG GTGAGGAAGA GGTAACAGTA ACTCTGACAG GTCACACGACATGACCGCGA CGCTGTGTGC ACCCGCTCCG TCACCGTCCG GCTGCCTGGCCTGCACAACA GCCTTGTGAA ACTGAAGCAT GGGGCAGGAG TTGCCATGGATACTGGCGCT GCGACACACG TGGGCGAGGC AGTGGCAGGC CGACGGACCGGACGTGTTGT CGGAACACTT TGACTTCGTA CCCCGTCCTC AACGGTACCTTGGCCAGGAC ATCCAGCTCC CCCTCCTGAA AGGTGACCTC CGCATCCAGCATACAGTGAC GGCCTCCGTG CGCCTCAGCT ACGGGGAGGA CCTGCAGATGACCGGTCCTG TAGGTCGAGG GGGAGGACTT TCCACTGGAG GCGTAGGTCGTATGTCACTG CCGGAGGCAC GCGGAGTCGA TGCCCCTCCT GGACGTCTACGACTGGGATG GCCGCGGGAG GCTGCTGGTG AAGCTGTCCC CCGTCTATGCCGGGAAGACC TGCGGCCTGT GTGGGAATTA CAATGGCAAC CAGGGCGACGCTGACCCTAC CGGCGCCCTC CGACGACCAC TTCGACAGGG GGCAGATACGGCCCTTCTGG ACGCCGGACA CACCCTTAAT GTTACCGTTG GTCCCGCTGCACTTCCTTAC CCCCTCTGGG CTGGCRGAGC CCCGGGTGGA GGACTTCGGGAACGCCTGGA AGCTGCACGG GGACTGCCAG GACCTGCAGA AGCAGCACAGTGAAGGAATG GGGGAGACCC GACCGYCTCG GGGCCCACCT CCTGAAGCCCTTGCGGACCT TCGACGTGCC CCTGACGGTC CTGGACGTCT TCGTCGTGTCCGATCCCTGC GCCCTCAACC CGCGCATGAC CAGGTTCTCC GAGGAGGCGTGCGCGGTCCT GACGTCCCCC ACATTCGAGG CCTGCCATCG TGCCGTCAGCGCTAGGGACG CGGGAGTTGG GCGCGTACTG GTCCAAGAGG CTCCTCCGCACGCGCCAGGA CTGCAGGGGG TGTAAGCTCC GGACGGTAGC ACGGCAGTCGCCGCTGCCCT ACCTGCGGAA CTGCCGCTAC GACGTGTGCT CCTGCTCGGACGGCCGCGAG TGCCTGTGCG GCGCCCTGGC CAGCTATGCC GCGGCCTGCGGGCGACGGGA TGGACGCCTT GACGGCGATG CTGCACACGA GGACGAGCCTGCCGGCGCTC ACGGACACGC CGCGGGACCG GTCGATACGG CGCCGGACGCCGGGGAGAGG CGTGCGCGTC GCGTGGCGCG AGCCAGGCCG CTGTGAGCTGAACTGCCCGA AAGGCCAGGT GTACCTGCAG TGCGGGACCC CCTGCAACCTGCCCCTCTCC GCACGCGCAG CGCACCGCGC TCGGTCCGGC GACACTCGACTTGACGGGCT TTCCGGTCCA CATGGACGTC ACGCCCTGGG GGACGTTGGAGACCTGCCGC TCTCTCTCTT ACCCGGATGA GGAATGCAAT GAGGCCTGCCTGGAGGGCTG CTTCTGCCCC CCAGGGCTCT ACATGGATGA GAGGGGGGACCTGGACGGCG AGAGAGAGAA TGGGCCTACT CCTTACGTTA CTCCGGACGGACCTCCCGAC GAAGACGGGG GGTCCCGAGA TGTACCTACT CTCCCCCCTGTGCGTGCCCA AGGCCCAGTG CCCCTGTTAC TATGACGGTG AGATCTTCCAGCCAGAAGAC ATCTTCTCAG ACCATCACAC CATGTGCTAC TGTGAGGATGACGCACGGGT TCCGGGTCAC GGGGACAATG ATACTGCCAC TCTAGAAGGTCGGTCTTCTG TAGAAGAGTC TGGTAGTGTG GTACACGATG ACACTCCTACGCTTCATGCA CTGTACCATG AGTGGAGTCC CCGGAAGCTT GCTGCCTGACGCTGTCCTCA GCAGTCCCCT GTCTCATCGC AGCAAAAGGA GCCTATCCTGCGAAGTACGT GACATGGTAC TCACCTCAGG GGCCTTCGAA CGACGGACTGCGACAGGAGT CGTCAGGGGA CAGAGTAGCG TCGTTTTCCT CGGATAGGACTCGGCCCCCC ATGGTCAAGC TGGTGTGTCC CGCTGACAAC CTGCGGGCTGAAGGGCTCGA GTGTACCAAA ACGTGCCAGA ACTATGACCT GGAGTGCATGAGCCGGGGGG TACCAGTTCG ACCACACAGG GCGACTGTTG GACGCCCGACTTCCCGAGCT CACATGGTTT TGCACGGTCT TGATACTGGA CCTCACGTACAGCATGGGCT GTGTCTCTGG CTGCCTCTGC CCCCCGGGCA TGGTCCGGCATGAGAACAGA TGTGTGGCCC TGGAAAGGTG TCCCTGCTTC CATCAGGGCATCGTACCCGA CACAGAGACC GACGGAGACG GGGGGCCCGT ACCAGGCCGTACTCTTGTCT ACACACCGGG ACCTTTCCAC AGGGACGAAG GTAGTCCCGTAGGAGTATGC CCCTGGAGAA ACAGTGAAGA TTGGCTGCAA CACTTGTGTCTGTCGGGACC GGAAGTGGAA CTGCACAGAC CATGTGTGTG ATGCCACGTGTCCTCATACG GGGACCTCTT TGTCACTTCT AACCGACGTT GTGAACACAGACAGCCCTGG CCTTCACCTT GACGTGTCTG GTACACACAC TACGGTGCACCTCCACGATC GGCATGGCCC ACTACCTCAC CTTCGACGGG CTCAAATACCTGTTCCCCGG GGAGTGCCAG TACGTTCTGG TGCAGGATTA CTGCGGCAGTGAGGTGCTAG CCGTACCGGG TGATGGAGTG GAAGCTGCCC GAGTTTATGGACAAGGGGCC CCTCACGGTC ATGCAAGACC ACGTCCTAAT GACGCCGTCAAACCCTGGGA CCTTTCGGAT CCTAGTGGGG AATAAGGGAT GCAGCCACCCCTCAGTGAAA TGCAAGAAAC GGGTCACCAT CCTGGTGGAG GGAGGAGAGATTGGGACCCT GGAAAGCCTA GGATCACCCC TTATTCCCTA CGTCGGTGGGGAGTCACTTT ACGTTCTTTG CCCAGTGGTA GGACCACCTC CCTCCTCTCTTTGAGCTGTT TGACGGGGAG GTGAATGTGA AGAGGCCCAT GAAGGATGAGACTCACTTTG AGGTGGTGGA GTCTGGCCGG TACATCATTC TGCTGCTGGGAACTCGACAA ACTGCCCCTC CACTTACACT TCTCCGGGTA CTTCCTACTCTGAGTGAAAC TCCACCACCT CAGACCGGCC ATGTAGTAAG ACGACGACCCCAAAGCCCTC TCCGTGGTCT GGGACCGCCA CCTGAGCATC TCCGTGGTCCTGAAGCAGAC ATACCAGGAG AAAGTGTGTG GCCTGTGTGG GAATTTTGATGTTTCGGGAG AGGCACCAGA CCCTGGCGGT GGACTCGTAG AGGCACCAGGACTTCGTCTG TATGGTCCTC TTTCACACAC CGGACACACC CTTAAAACTAGGCATCCAGA ACAATGACCT CACCAGCAGC AACCTCCAAG TGGAGGAAGACCCTGTGGAC TTTGGGAACT CCTGGAAAGT GAGCTCGCAG TGTGCTGACACCGTAGGTCT TGTTACTGGA GTGGTCGTCG TTGGAGGTTC ACCTCCTTCTGGGACACCTG AAACCCTTGA GGACCTTTCA CTCGAGCGTC ACACGACTGTCCAGAAAAGT GCCTCTGGAC TCATCCCCTG CCACCTGCCA TAACAACATCATGAAGCAGA CGATGGTGGA TTCCTCCTGT AGAATCCTTA CCAGTGACGTGGTCTTTTCA CGGAGACCTG AGTAGGGGAC GGTGGACGGT ATTGTTGTAGTACTTCGTCT GCTACCACCT AAGGAGGACA TCTTAGGAAT GGTCACTGCACTTCCAGGAC TGCAACAAGC TGGTGGACCC CGAGCCATAT CTGGATGTCTGCATTTACGA CACCTGCTCC TGTGAGTCCA TTGGGGACTG CGCCTGCTTCGAAGGTCCTG ACGTTGTTCG ACCACCTGGG GCTCGGTATA GACCTACAGACGTAAATGCT GTGGACGAGG ACACTCAGGT AACCCCTGAC GCGGACGAAGTGCGACACCA TTGCTGCCTA TGCCCACGTG TGTGCCCAGC ATGGCAAGGTGGTGACCTGG AGGACGGCCA CATTGTGCCC CCAGAGCTGC GAGGAGAGGAACGCTGTGGT AACGACGGAT ACGGGTGCAC ACACGGGTCG TACCGTTCCACCACTGGACC TCCTGCCGGT GTAACACGGG GGTCTCGACG CTCCTCTCCTATCTCCGGGA GAACGGGTAT GAGTGTGAGT GGCGCTATAA CAGCTGTGCACCTGCCTGTC AAGTCACGTG TCAGCACCCT GAGCCACTGG CCTGCCCTGTTAGAGGCCCT CTTGCCCATA CTCACACTCA CCGCGATATT GTCGACACGTGGACGGACAG TTCAGTGCAC AGTCGTGGGA CTCGGTGACC GGACGGGACAGCAGTGTGTG GAGGGCTGCC ATGCCCACTG CCCTCCAGGG AAAATCCTGGATGAGCTTTT GCAGACCTGC GTTGACCCTG AAGACTGTCC AGTGTGTGAGCGTCACACAC CTCCCGACGG TACGGGTGAC GGGAGGTCCC TTTTAGGACCTACTCGAAAA CGTCTGGACG CAACTGGGAC TTCTGACAGG TCACACACTCGTGGCTGGCC GGCGTTTTGC CTCAGGAAAG AAAGTCACCT TGAATCCCAGTGACCCTGAG CACTGCCAGA TTTGCCACTG TGATGTTGTC AACCTCACCTCACCGACCGG CCGCAAAACG GAGTCCTTTC TTTCAGTGGA ACTTAGGGTCACTGGGACTC GTGACGGTCT AAACGGTGAC ACTACAACAG TTGGAGTGGAGTGAAGCCTG CCAGGAGCCG GGAGGCCTGG TGGTGCCTCC CACAGATGCCCCGGTGAGCC CCACCACTCT GTATGTGGAG GACATCTCGG AACCGCCGTTCACTTCGGAC GGTCCTCGGC CCTCCGGACC ACCACGGAGG GTGTCTACGGGGCCACTCGG GGTGGTGAGA CATACACCTC CTGTAGAGCC TTGGCGGCAAGCACGATTTC TACTGCAGCA GGCTACTGGA CCTGGTCTTC CTGCTGGATGGCTCCTCCAG GCTGTCCGAG GCTGAGTTTG AAGTGCTGAA GGCCTTTGTGCGTGCTAAAG ATGACGTCGT CCGATGACCT GGACCAGAAG GACGACCTACCGAGGAGGTC CGACAGGCTC CGACTCAAAC TTCACGACTT CCGGAAACACGTGGACATGA TGGAGCGGCT GCGCATCTCC CAGAAGTGGG TCCGCGTGGCCGTGGTGGAG TACCACGACG GCTCCCACGC CTACATCGGG CTCAAGGACCCACCTGTACT ACCTCGCCGA CGCGTAGAGG GTCTTCACCC AGGCGCACCGGCACCACCTC ATGGTGCTGC CGAGGGTGCG GATGTAGCCC GAGTTCCTGGGGAAGCGACC GTCAGAGCTG CGGCGCATTG CCAGCCAGGT GAAGTATGCGGGCAGCCAGG TGGCCTCCAC CAGCGAGGTC TTGAAATACA CACTGTTCCACCTTCGCTGG CAGTCTCGAC GCCGCGTAAC GGTCGGTCCA CTTCATACGCCCGTCGGTCC ACCGGAGGTG GTCGCTCCAG AACTTTATGT GTGACAAGGTAATCTTCAGC AAGATCGACC GCCCTGAAGC CTCCCGCATC GCCCTGCTCCTGATGGCCAG CCAGGAGCCC CAACGGATGT CCCGGAACTT TGTCCGCTACTTAGAAGTCG TTCTAGCTGG CGGGACTTCG GAGGGCGTAG CGGGACGAGGACTACCGGTC GGTCCTCGGG GTTGCCTACA GGGCCTTGAA ACAGGCGATGGTCCAGGGCC TGAAGAAGAA GAAGGTCATT GTGATCCCGG TGGGCATTGGGCCCCATGCC AACCTCAAGC AGATCCGCCT CATCGAGAAG CAGGCCCCTGCAGGTCCCGG ACTTCTTCTT CTTCCAGTAA CACTAGGGCC ACCCGTAACCCGGGGTACGG TTGGAGTTCG TCTAGGCGGA GTAGCTCTTC GTCCGGGGACAGAACAAGGC CTTCGTGCTG AGCAGTGTGG ATGAGCTGGA GCAGCAAAGGGACGAGATCG TTAGCTACCT CTGTGACCTT GCCCCTGAAG CCCCTCCTCCTCTTGTTCCG GAAGCACGAC TCGTCACACC TACTCGACCT CGTCGTTTCCCTGCTCTAGC AATCGATGGA GACACTGGAA CGGGGACTTC GGGGAGGAGGTACTCTGCCC CCCGACATGG CACAAGTCAC TGTGGGCCCG GGGCTCTTGGGGGTTTCGAC CCTGGGGCCC AAGAGGAACT CCATGGTTCT GGATGTGGCGATGAGACGGG GGGCTGTACC GTGTTCAGTG ACACCCGGGC CCCGAGAACCCCCAAAGCTG GGACCCCGGG TTCTCCTTGA GGTACCAAGA CCTACACCGCTTCGTCCTGG AAGGATCGGA CAAAATTGGT GAAGCCGACT TCAACAGGAGCAAGGAGTTC ATGGAGGAGG TGATTCAGCG GATGGATGTG GGCCAGGACAAAGCAGGACC TTCCTAGCCT GTTTTAACCA CTTCGGCTGA AGTTGTCCTCGTTCCTCAAG TACCTCCTCC ACTAAGTCGC CTACCTACAC CCGGTCCTGTGCATCCACGT CACGGTGCTG CAGTACTCCT ACATGGTGAC CGTGGAGTACCCCTTCAGCG AGGCACAGTC CAAAGGGGAC ATCCTGCAGC GGGTGCGAGACGTAGGTGCA GTGCCACGAC GTCATGAGGA TGTACCACTG GCACCTCATGGGGAAGTCGC TCCGTGTCAG GTTTCCCCTG TAGGACGTCG CCCACGCTCTGATCCGCTAC CAGGGCGGCA ACAGGACCAA CACTGGGCTG GCCCTGCGGTACCTCTCTGA CCACAGCTTC TTGGTCAGCC AGGGTGACCG GGAGCAGGCGCTAGGCGATG GTCCCGCCGT TGTCCTGGTT GTGACCCGAC CGGGACGCCATGGAGAGACT GGTGTCGAAG AACCAGTCGG TCCCACTGGC CCTCGTCCGCCCCAACCTGG TCTACATGGT CACCGGAAAT CCTGCCTCTG ATGAGATCAAGAGGCTGCCT GGAGACATCC AGGTGGTGCC CATTGGAGTG GGCCCTAATGGGGTTGGACC AGATGTACCA GTGGCCTTTA GGACGGAGAC TACTCTAGTTCTCCGACGGA CCTCTGTAGG TCCACCACGG GTAACCTCAC CCGGGATTACCCAACGTGCA GGAGCTGGAG AGGATTGGCT GGCCCAATGC CCCTATCCTCATCCAGGACT TTGAGACGCT CCCCCGAGAG GCTCCTGACC TGGTGCTGCAGGTTGCACGT CCTCGACCTC TCCTAACCGA CCGGGTTACG GGGATAGGAGTAGGTCCTGA AACTCTGCGA GGGGGCTCTC CGAGGACTGG ACCACGACGTGAGGTGCTGC TCCGGAGAGG GGCTGCAGAT CCCCACCCTC TCCCCTGCACCTGACTGCAG CCAGCCCCTG GACGTGATCC TTCTCCTGGA TGGCTCCTCCCTCCACGACG AGGCCTCTCC CCGACGTCTA GGGGTGGGAG AGGGGACGTGGACTGACGTC GGTCGGGGAC CTGCACTAGG AAGAGGACCT ACCGAGGAGGAGTTTCCCAG CTTCTTATTT TGATGAAATG AAGAGTTTCG CCAAGGCTTTCATTTCAAAA GCCAATATAG GGCCTCGTCT CACTCAGGTG TCAGTGCTGCTCAAAGGGTC GAAGAATAAA ACTACTTTAC TTCTCAAAGC GGTTCCGAAAGTAAAGTTTT CGGTTATATC CCGGAGCAGA GTGAGTCCAC AGTCACGACGAGTATGGAAG CATCACCACC ATTGACGTGC CATGGAACGT GGTCCCGGAGAAAGCCCATT TGCTGAGCCT TGTGGACGTC ATGCAGCGGG AGGGAGGCCCTCATACCTTC GTAGTGGTGG TAACTGCACG GTACCTTGCA CCAGGGCCTCTTTCGGGTAA ACGACTCGGA ACACCTGCAG TACGTCGCCC TCCCTCCGGGCAGCCAAATC GGGGATGCCT TGGGCTTTGC TGTGCGATAC TTGACTTCAGAAATGCATGG TGCCAGGCCG GGAGCCTCAA AGGCGGTGGT CATCCTGGTCGTCGGTTTAG CCCCTACGGA ACCCGAAACG ACACGCTATG AACTGAAGTCTTTACGTACC ACGGTCCGGC CCTCGGAGTT TCCGCCACCA GTAGGACCAGACGGACGTCT CTGTGGATTC AGTGGATGCA GCAGCTGATG CCGCCAGGTCCAACAGAGTG ACAGTGTTCC CTATTGGAAT TGGAGATCGC TACGATGCAGTGCCTGCAGA GACACCTAAG TCACCTACGT CGTCGACTAC GGCGGTCCAGGTTGTCTCAC TGTCACAAGG GATAACCTTA ACCTCTAGCG ATGCTACGTCCCCAGCTACG GATCTTGGCA GGCCCAGCAG GCGACTCCAA CGTGGTGAAGCTCCAGCGAA TCGAAGACCT CCCTACCATG GTCACCTTGG GCAATTCCTTGGGTCGATGC CTAGAACCGT CCGGGTCGTC CGCTGAGGTT GCACCACTTCGAGGTCGCTT AGCTTCTGGA GGGATGGTAC CAGTGGAACC CGTTAAGGAACCTCCACAAA CTGTGCTCTG GATTTGTTAG GATTTGCATG GATGAGGATGGGAATGAGAA GAGGCCCGGG GACGTCTGGA CCTTGCCAGA CCAGTGCCACGGAGGTGTTT GACACGAGAC CTAAACAATC CTAAACGTAC CTACTCCTACCCTTACTCTT CTCCGGGCCC CTGCAGACCT GGAACGGTCT GGTCACGGTGACCGTGACTT GCCAGCCAGA TGGCCAGACC TTGCTGAAGA GTCATCGGGTCAACTGTGAC CGGGGGCTGA GGCCTTCGTG CCCTAACAGC CAGTCCCCTGTGGCACTGAA CGGTCGGTCT ACCGGTCTGG AACGACTTCT CAGTAGCCCAGTTGACACTG GCCCCCGACT CCGGAAGCAC GGGATTGTCG GTCAGGGGACTTAAAGTGGA AGAGACCTGT GGCTGCCGCT GGACCTGCCC CTGYGTGTGCACAGGCAGCT CCACTCGGCA CATCGTGACC TTTGATGGGC AGAATTTCAAAATTTCACCT TCTCTGGACA CCGACGGCGA CCTGGACGGG GACRCACACGTGTCCGTCGA GGTGAGCCGT GTAGCACTGG AAACTACCCG TCTTAAAGTTGCTGACTGGC AGCTGTTCTT ATGTCCTATT TCAAAACAAG GAGCAGGACCTGGAGGTGAT TCTCCATAAT GGTGCCTGCA GCCCTGGAGC AAGGCAGGGCCGACTGACCG TCGACAAGAA TACAGGATAA AGTTTTGTTC CTCGTCCTGGACCTCCACTA AGAGGTATTA CCACGGACGT CGGGACCTCG TTCCGTCCCGTGCATGAAAT CCATCGAGGT GAAGCACAGT GCCCTCTCCG TCGAGSTGCACAGTGACATG GAGGTGACGG TGAATGGGAG ACTGGTCTCT GTTCCTTACGACGTACTTTA GGTAGCTCCA CTTCGTGTCA CGGGAGAGGC AGCTCSACGTGTCACTGTAC CTCCACTGCC ACTTACCCTC TGACCAGAGA CAAGGAATGCTGGGTGGGAA CATGGAAGTC AACGTTTATG GTGCCATCAT GCATGAGGTCAGATTCAATC ACCTTGGTCA CATCTTCACA TTCACTCCAC AAAACAATGAACCCACCCTT GTACCTTCAG TTGCAAATAC CACGGTAGTA CGTACTCCAGTCTAAGTTAG TGGAACCAGT GTAGAAGTGT AAGTGAGGTG TTTTGTTACTGTTCCAACTG CAGCTCAGCC CCAAGACTTT TGCTTCAAAG ACGTATGGTCTGTGTGGGAT CTGTGATGAG AACGGAGCCA ATGACTTCAT GCTGAGGGATCAAGGTTGAC GTCGAGTCGG GGTTCTGAAA ACGAAGTTTC TGCATACCAGACACACCCTA GACACTACTC TTGCCTCGGT TACTGAAGTA CGACTCCCTAGGCACAGTCA CCACAGACTG GAAAACACTT GTTCAGGAAT GGACTGTGCAGCGGCCAGGG CAGACGTGCC AGCCCATCCT GGAGGAGCAG TGTCTTGTCCCCGTGTCAGT GGTGTCTGAC CTTTTGTGAA CAAGTCCTTA CCTGACACGTCGCCGGTCCC GTCTGCACGG TCGGGTAGGA CCTCCTCGTC ACAGAACAGGCCGACAGCTC CCACTGCCAG GTCCTCCTCT TACCACTGTT TGCTGAATGCCACAAGGTCC TGGCTCCAGC CACATTCTAT GCCATCTGCC AGCAGGACAGGGCTGTCGAG GGTGACGGTC CAGGAGGAGA ATGGTGACAA ACGACTTACGGTGTTCCAGG ACCGAGGTCG GTGTAAGATA CGGTAGACGG TCGTCCTGTCTTGCCACCAG GAGCAAGTGT GTGAGGTGAT CGCCTCTTAT GCCCACCTCTGTCGGACCAA CGGGGTCTGC GTTGACTGGA GGACACCTGA TTTCTGTGCTAACGGTGGTC CTCGTTCACA CACTCCACTA GCGGAGAATA CGGGTGGAGACAGCCTGGTT GCCCCAGACG CAACTGACCT CCTGTGGACT AAAGACACGAATGTCATGCC CACCATCTCT GGTCTACAAC CACTGTGAGC ATGGCTGTCCCCGGCACTGT GATGGCAACG TGAGCTCCTG TGGGGACCAT CCCTCCGAAGTACAGTACGG GTGGTAGAGA CCAGATGTTG GTGACACTCG TACCGACAGGGGCCGTGACA CTACCGTTGC ACTCGAGGAC ACCCCTGGTA GGGAGGCTTCGCTGTTTCTG CCCTCCAGAT AAAGTCATGT TGGAAGGCAG CTGTGTCCCTGAAGAGGCCT GCACTCAGTG CATTGGTGAG GATGGAGTCC AGCACCAGTTCGACAAAGAC GGGAGGTCTA TTTCAGTACA ACCTTCCGTC GACACAGGGACTTCTCCGGA CGTGAGTCAC GTAACCACTC CTACCTCAGG TCGTGGTCAACCTGGAAGCC TGGGTCCCGG ACCACCAGCC CTGTCAGATC TGCACATGCCTCAGCGGGCG GAAGGTCAAC TGCACAACGC AGCCCTGCCC CACGGCCAAAGGACCTTCGG ACCCAGGGCC TGGTGGTCGG GACAGTCTAG ACGTGTACGGAGTCGCCCGC CTTCCAGTTG ACGTGTTGCG TCGGGACGGG GTGCCGGTTTGCTCCCACGT GTGGCCTGTG TGAAGTAGCC CGCCTCCGCC AGAATGCAGACCAGTGCTGC CCCGAGTATG AGTGTGTGTG TGACCCAGTG AGCTGTGACCCGAGGGTGCA CACCGGACAC ACTTCATCGG GCGGAGGCGG TCTTACGTCTGGTCACGACG GGGCTCATAC TCACACACAC ACTGGGTCAC TCGACACTGGTGCCCCCAGT GCCTCACTGT GAACGTGGCC TCCAGCCCAC ACTGACCAACCCTGGCGAGT GCAGACCCAA CTTCACCTGC GCCTGCAGGA AGGAGGAGTGACGGGGGTCA CGGAGTGACA CTTGCACCGG AGGTCGGGTG TGACTGGTTGGGACCGCTCA CGTCTGGGTT GAAGTGGACG CGGACGTCCT TCCTCCTCACCAAAAGAGTG TCCCCACCCT CCTGCCCCCC GCACCGTTTG CCCACCCTTCGGAAGACCCA GTGCTGTGAT GAGTATGAGT GTGCCTGCAA CTGTGTCAACGTTTTCTCAC AGGGGTGGGA GGACGGGGGG CGTGGCAAAC GGGTGGGAAGCCTTCTGGGT CACGACACTA CTCATACTCA CACGGACGTT GACACAGTTGTCCACAGTGA GCTGTCCCCT TGGGTACTTG GCCTCAACCG CCACCAATGACTGTGGCTGT ACCACAACCA CCTGCCTTCC CGACAAGGTG TGTGTCCACCAGGTGTCACT CGACAGGGGA ACCCATGAAC CGGAGTTGGC GGTGGTTACTGACACCGACA TGGTGTTGGT GGACGGAAGG GCTGTTCCAC ACACAGGTGGGAAGCACCAT CTACCCTGTG GGCCAGTTCT GGGAGGAGGG CTGCGATGTGTGCACCTGCA CCGACATGGA GGATGCCGTG ATGGGCCTCC GCGTGGCCCACTTCGTGGTA GATGGGACAC CCGGTCAAGA CCCTCCTCCC GACGCTACACACGTGGACGT GGCTGTACCT CCTACGGCAC TACCCGGAGG CGCACCGGGTGTGCTCCCAG AAGCCCTGTG AGGACAGCTG TCGGTCGGGC TTCACTTACGTTCTGCATGA AGGCGAGTGC TGTGGAAGGT GCCTGCCATC TGCCTGTGAGCACGAGGGTC TTCGGGACAC TCCTGTCGAC AGCCAGCCCG AAGTGAATGCAAGACGTACT TCCGCTCACG ACACCTTCCA CGGACGGTAG ACGGACACTCGTGGTGACTG GCTCACCGCG GGGGGACTCC CAGTCTTCCT GGAAGAGTGTCGGCTCCCAG TGGGCCTCCC CGGAGAACCC CTGCCTCATC AATGAGTGTGCACCACTGAC CGAGTGGCGC CCCCCTGAGG GTCAGAAGGA CCTTCTCACAGCCGAGGGTC ACCCGGAGGG GCCTCTTGGG GACGGAGTAG TTACTCACACTCCGAGTGAA GGAGGAGGTC TTTATACAAC AAAGGAACGT CTCCTGCCCCCAGCTGGAGG TCCCTGTCTG CCCCTCGGGC TTTCAGCTGA GCTGTAAGACAGGCTCACTT CCTCCTCCAG AAATATGTTG TTTCCTTGCA GAGGACGGGGGTCGACCTCC AGGGACAGAC GGGGAGCCCG AAAGTCGACT CGACATTCTGCTCAGCGTGC TGCCCAAGCT GTCGCTGTGA GCGCATGGAG GCCTGCATGCTCAATGGCAC TGTCATTGGG CCCGGGAAGA CTGTGATGAT CGATGTGTGCGAGTCGCACG ACGGGTTCGA CAGCGACACT CGCGTACCTC CGGACGTACGAGTTACCGTG ACAGTAACCC GGGCCCTTCT GACACTACTA GCTACACACGACGACCTGCC GCTGCATGGT GCAGGTGGGG GTCATCTCTG GATTCAAGCTGGAGTGCAGG AAGACCACCT GCAACCCCTG CCCCCTGGGT TACAAGGAAGTGCTGGACGG CGACGTACCA CGTCCACCCC CAGTAGAGAC CTAAGTTCGACCTCACGTCC TTCTGGTGGA CGTTGGGGAC GGGGGACCCA ATGTTCCTTCAAAATAACAC AGGTGAATGT TGTGGGAGAT GTTTGCCTAC GGCTTGCACCATTCAGCTAA GAGGAGGACA GATCATGACA CTGAAGCGTG ATGAGACGCTTTTTATTGTG TCCACTTACA ACACCCTCTA CAAACGGATG CCGAACGTGGTAAGTCGATT CTCCTCCTGT CTAGTACTGT GACTTCGCAC TACTCTGCGACCAGGATGGC TGTGATACTC ACTTCTGCAA GGTCAATGAG AGAGGAGAGTACTTCTGGGA GAAGAGGGTC ACAGGCTGCC CACCCTTTGA TGAACACAAGGGTCCTACCG ACACTATGAG TGAAGACGTT CCAGTTACTC TCTCCTCTCATGAAGACCCT CTTCTCCCAG TGTCCGACGG GTGGGAAACT ACTTGTGTTCTGTCTTGCTG AGGGAGGTAA AATTATGAAA ATTCCAGGCA CCTGCTGTGACACATGTGAG GAGCCTGAGT GCAACGACAT CACTGCCAGG CTGCAGTATGACAGAACGAC TCCCTCCATT TTAATACTTT TAAGGTCCGT GGACGACACTGTGTACACTC CTCGGACTCA CGTTGCTGTA GTGACGGTCC GACGTCATACTCAAGGTGGG AAGCTGTAAG TCTGAAGTAG AGGTGGATAT CCACTACTGCCAGGGCAAAT GTGCCAGCAA AGCCATGTAC TCCATTGACA TCAACGATGTAGTTCCACCC TTCGACATTC AGACTTCATC TCCACCTATA GGTGATGACGGTCCCGTTTA CACGGTCGTT TCGGTACATG AGGTAACTGT AGTTGCTACAGCAGGACCAG TGCTCCTGCT GCTCTCCGAC ACGGACGGAG CCCATGCAGGTGGCCCTGCA CTGCACCAAT GGCTCTGTTG TGTACCATGA GGTTCTCAATCGTCCTGGTC ACGAGGACGA CGAGAGGCTG TGCCTGCCTC GGGTACGTCCACCGGGACGT GACGTGGTTA CCGAGACAAC ACATGGTACT CCAAGAGTTAGCCATGGAGT GCAAATGCTC CCCCAGGAAG TGCAGCAAGT GA

The present invention is directed to a von Willebrand Factor (VWF)fragment comprising a D′ domain and a D3 domain of VWF, wherein the VWFfragment inhibits binding of endogenous VWF (full-length VWF) to a FVIIIprotein. In one embodiment, the VWF fragment binds to or is associatedwith a FVIII protein. By binding to or associating with a FVIII protein,a VWF fragment of the invention protects FVIII from protease cleavageand FVIII activation, stabilizes the heavy chain and light chain ofFVIII, and prevents clearance of FVIII by scavenger receptors. Inanother embodiment, the VWF fragment binds to or associates with a FVIIIprotein and blocks or prevents binding of the FVIII protein tophospholipid and activated Protein C. By preventing or inhibitingbinding of the FVIII protein with endogenous, full-length VWF, the VWFfragment of the invention reduces the clearance of FVIII by VWFclearance receptors and thus extends the half-life of FVIII. Thehalf-life extension of a FVIII protein is thus due to the binding of orassociating with the VWF fragment lacking a VWF clearance receptorbinding site to the FVIII protein and shielding or protecting of theFVIII protein by the VWF fragment from endogenous VWF which contains theVWF clearance receptor binding site. The FVIII protein bound to orprotected by the VWF fragment can also allow recycling of a FVIIIprotein. Therefore, the VWF fragment cannot be full-length mature VWF.By eliminating the VWF clearance pathway receptor binding sitescontained in the full length VWF molecule, the FVIII/VWF heterodimers ofthe invention are uncoupled from the VWF clearance pathway, which allowsthe further extending FVIII half-life.

The VWF fragment comprising the D′ domain and the D3 domain can furthercomprise a VWF domain selected from the group consisting of an A1domain, an A2 domain, an A3 domain, a D1 domain, a D2 domain, a D4domain, a B1 domain, a B2 domain, a B3 domain, a C1 domain, a C2 domain,a CK domain, one or more fragments thereof, and any combinationsthereof. In one embodiment, a VWF fragment comprises, consistsessentially of, or consists of: (1) the D′ and D3 domains of VWF orfragments thereof (2) the D1, D′, and D3 domains of VWF or fragmentsthereof (3) the D2, D′, and D3 domains of VWF or fragments thereof; (4)the D1, D2, D′, and D3 domains of VWF or fragments thereof; or (5) theD1, D2, D′, D3, and A1 domains of VWF or fragments thereof. The VWFfragment described herein does not contain a site binding to a VWFclearance receptor. In another embodiment, the VWF fragment describedherein is not amino acids 764 to 1274 of SEQ ID NO: 2. The VWF fragmentof the present invention can comprise any other sequences linked to orfused to the VWF fragment, but is not the full-length VWF. For example,a VWF fragment described herein can further comprise a signal peptide.

In one embodiment, a VWF fragment of the present invention comprises theD′ domain and the D3 domain of VWF, wherein the D′ domain is at least60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical toamino acids 764 to 866 of SEQ ID NO: 2, wherein the VWF fragment bindsto a FVIII protein, shields, inhibits or prevents binding of endogenousVWF fragment to a FVIII protein. In another embodiment, a VWF fragmentcomprises the D′ domain and the D3 domain of VWF, wherein the D3 domainis at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to amino acids 867 to 1240 of SEQ ID NO: 2, wherein the VWFfragment binds to a FVIII protein or inhibits or prevents binding ofendogenous VWF fragment to a FVIII protein. In some embodiments, a VWFfragment described herein comprises, consists essentially of, orconsists of the D′ domain and D3 domain of VWF, which are at least 60%,70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to aminoacids 764 to 1240 of SEQ ID NO: 2, wherein the VWF fragment binds to aFVIII protein or inhibits or prevents binding of endogenous VWF fragmentto a FVIII protein. In other embodiments, a VWF fragment comprises,consists essentially of, or consists of the D1, D2′, D, and D3 domainsat least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to amino acids 23 to 1240 of SEQ ID NO: 2, wherein the VWFfragment binds to a FVIII protein or inhibits or prevents binding ofendogenous VWF fragment to a FVIII protein. In still other embodiments,the VWF fragment further comprises a signal peptide operably linkedthereto.

In some embodiments, a VWF fragment of the invention consistsessentially of or consists of (1) the D′D3 domain, the D1D′D3 domain,D2D′D3 domain, or D1D2D′D3 domain and (2) an additional VWF sequence upto about 10 amino acids (e.g., any sequences from amino acids 764 to1240 of SEQ ID NO: 2 to amino acids 764 to 1250 of SEQ ID NO: 2), up toabout 15 amino acids (e.g., any sequences from amino acids 764 to 1240of SEQ ID NO: 2 to amino acids 764 to 1255 of SEQ ID NO: 2), up to about20 amino acids (e.g., any sequences from amino acids 764 to 1240 of SEQID NO: 2 to amino acids 764 to 1260 of SEQ ID NO: 2), up to about 25amino acids (e.g., any sequences from amino acids 764 to 1240 of SEQ IDNO: 2 to amino acids 764 to 1265 of SEQ ID NO: 2), or up to about 30amino acids (e.g., any sequences from amino acids 764 to 1240 of SEQ IDNO: 2 to amino acids 764 to 1260 of SEQ ID NO: 2). In a particularembodiment, the VWF fragment comprising or consisting essentially of theD′ domain and the D3 domain is neither amino acids 764 to 1274 of SEQ IDNO: 2 nor the full-length mature VWF.

In other embodiments, the VWF fragment comprising the D′D3 domainslinked to the D1D2 domains further comprises an intracellular cleavagesite, e.g., (a cleavage site by PACE or PC5), allowing cleavage of theD1D2 domains from the D′D3 domains upon expression. Non-limitingexamples of the intracellular cleavage site are disclosed elsewhereherein.

In yet other embodiments, a VWF fragment comprises the D′ domain and theD3 domain, but does not comprise an amino acid sequence selected fromthe group consisting of (1) amino acids 1241 to 2813 of SEQ ID NO: 2,(2) amino acids 1270 to amino acids 2813 of SEQ ID NO: 2, (3) aminoacids 1271 to amino acids 2813 of SEQ ID NO: 2, (4) amino acids 1272 toamino acids 2813 of SEQ ID NO: 2, (5) amino acids 1273 to amino acids2813 of SEQ ID NO: 2, and (6) amino acids 1274 to amino acids 2813 ofSEQ ID NO: 2.

In still other embodiments, a VWF fragment of the present inventioncomprises, consists essentially of, or consists of an amino acidsequence corresponding to the D′ domain, D3 domain, and A1 domain,wherein the amino acid sequence is at least 60%, 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acid 764 to1479 of SEQ ID NO: 2, wherein the VWF binds to FVIII. In a particularembodiment, the VWF fragment is not amino acids 764 to 1274 of SEQ IDNO: 2.

In some embodiments, a VWF fragment of the invention comprises the D′domain and the D3 domain, but does not comprise at least one VWF domainselected from the group consisting of (1) an A1 domain, (2) an A2domain, (3) an A3 domain, (4) a D4 domain, (5) a B1 domain, (6) a B2domain, (7) a B3 domain, (8) a C1 domain, (9) a C2 domain, (10) a CKdomain, (11) a CK domain and C2 domain, (12) a CK domain, a C2 domain,and a C1 domain, (13) a CK domain, a C2 domain, a C1 domain, a B3domain, (14) a CK domain, a C2 domain, a C1 domain, a B3 domain, a B2domain, (15) a CK domain, a C2 domain, a C1 domain, a B3 domain, a B2domain, and a B1 domain, (16) a CK domain, a C2 domain, a C1 domain, aB3 domain, a B2 domain, a B1 domain, and a D4 domain, (17) a CK domain,a C2 domain, a C1 domain, a B3 domain, a B2 domain, a B1 domain, a D4domain, and an A3 domain, (18) a CK domain, a C2 domain, a C1 domain, aB3 domain, a B2 domain, a B1 domain, a D4 domain, an A3 domain, and anA2 domain, (19) a CK domain, a C2 domain, a C1 domain, a B3 domain, a B2domain, a B1 domain, a D4 domain, an A3 domain, an A2 domain, and an A1domain, and (20) any combinations thereof.

In yet other embodiments, the VWF fragment comprises the D′D3 domainsand one or more domains or modules. Examples of such domains or modulesinclude, but are not limited to, the domains and modules disclosed inZhour et al., Blood published online Apr. 6, 2012: DOI10.1182/blood-2012-01-405134. For example, the VWF fragment can comprisethe D′D3 domain and one or more domains or modules selected from thegroup consisting of A1 domain, A2 domain, A3 domain, D4N module, VWD4module, C8-4 module, TIL-4 module, C1 module, C2 module, C3 module, C4module, C5 module, C5 module, C6 module, and any combinations thereof.

In still other embodiments, the VWF fragment is linked to a heterologousmoiety, wherein the heterologous moiety is linked to the N-terminus orthe C-terminus of the VWF fragment or inserted between two amino acidsin the VWF fragment. For example, the insertion sites for theheterologous moiety in the VWF fragment can be in the D′ domain, the D3domain, or both. The heterologous moiety can be a half-life extender.

In certain embodiments, a VWF fragment of the invention forms amultimer, e.g., dimer, trimer, tetramer, pentamer, hexamer, heptamer, orthe higher order multimers. In other embodiments, the VWF fragment is amonomer having only one VWF fragment. In some embodiments, the VWFfragment of the present invention can have one or more amino acidsubstitutions, deletions, additions, or modifications. In oneembodiment, the VWF fragment can include amino acid substitutions,deletions, additions, or modifications such that the VWF fragment is notcapable of forming a disulfide bond or forming a dimer or a multimer. Inanother embodiment, the amino acid substitution is within the D′ domainand the D3 domain. In a particular embodiment, a VWF fragment of theinvention contains at least one amino acid substitution at a residuecorresponding to residue 1099, residue 1142, or both residues 1099 and1142 of SEQ ID NO: 2. The at least one amino acid substitution can beany amino acids that are not occurring naturally in the wild type VWF.For example, the amino acid substitution can be any amino acids otherthan cysteine, e.g., Isoleucine, Alanine, Leucine, Asparagine, Lysine,Aspartic acid, Methionine, Phenylalanine, Glutamic acid, Threonine,Glutamine, Tryptophan, Glycine, Valine, Proline, Serine, Tyrosine,Arginine, or Histidine. In another example, the amino acid substitutionhas one or more amino acids that prevent or inhibit the VWF fragmentsfrom forming multimers.

In certain embodiments, the VWF fragment useful herein can be furthermodified to improve its interaction with FVIII, e.g., to improve bindingaffinity to FVIII. As a non-limiting example, the VWF fragment comprisesa serine residue at the residue corresponding to amino acid 764 of SEQID NO: 2 and a lysine residue at the residue corresponding to amino acid773 of SEQ ID NO: 2. Residues 764 and/or 773 can contribute to thebinding affinity of the VWF fragments to FVIII. In other embodiments,the VWF fragment can have other modifications, e.g., the fragment can bepegylated, glycosylated, hesylated, or polysialylated.

B) Heterologous Moieties

The heterologous moiety can be a heterologous polypeptide or aheterologous non-polypeptide moiety. In certain embodiments, theheterologous moiety is a half-life extending molecule which is known inthe art and comprises a polypeptide, a non-polypeptide moiety, or thecombination of both. The heterologous polypeptide moiety can comprise animmunoglobulin constant region or a portion thereof, albumin or afragment thereof, an albumin binding moiety, transferrin or a fragmentthereof, a PAS sequence, a HAP sequence, a derivative or variantthereof, or any combinations thereof. In some embodiments, thenon-polypeptide binding moiety comprises polyethylene glycol (PEG),polysialic acid, hydroxyethyl starch (HES), a derivative thereof, or anycombinations thereof. In certain embodiments, there can be one, two,three or more heterologous moieties, which can each be the same ordifferent molecules.

1) Immunoglobulin Constant Region or Portion Thereof

An immunoglobulin constant region is comprised of domains denoted CH(constant heavy) domains (CH1, CH2, etc.). Depending on the isotype,(i.e. IgG, IgM, IgA IgD, or IgE), the constant region can be comprisedof three or four CH domains. Some isotypes (e.g. IgG) constant regionsalso contain a hinge region. See Janeway et al. 2001, Immunobiology,Garland Publishing, N.Y., N.Y.

An immunoglobulin constant region or a portion thereof for producing thechimeric protein of the present invention may be obtained from a numberof different sources. In preferred embodiments, an immunoglobulinconstant region or a portion thereof is derived from a humanimmunoglobulin. It is understood, however, that the immunoglobulinconstant region or a portion thereof may be derived from animmunoglobulin of another mammalian species, including for example, arodent (e.g. a mouse, rat, rabbit, guinea pig) or non-human primate(e.g. chimpanzee, macaque) species. Moreover, the immunoglobulinconstant region or a portion thereof may be derived from anyimmunoglobulin class, including IgM, IgG, IgD, IgA and IgE, and anyimmunoglobulin isotype, including IgG1, IgG2, IgG3 and IgG4. In oneembodiment, the human isotype IgG1 is used.

A variety of the immunoglobulin constant region gene sequences (e.g.human constant region gene sequences) are available in the form ofpublicly accessible deposits. Constant region domains sequence can beselected having a particular effector function (or lacking a particulareffector function) or with a particular modification to reduceimmunogenicity. Many sequences of antibodies and antibody-encoding geneshave been published and suitable Ig constant region sequences (e.g.hinge, CH2, and/or CH3 sequences, or portions thereof) can be derivedfrom these sequences using art recognized techniques. The geneticmaterial obtained using any of the foregoing methods may then be alteredor synthesized to obtain polypeptides of the present invention. It willfurther be appreciated that the scope of this invention encompassesalleles, variants and mutations of constant region DNA sequences.

The sequences of the immunoglobulin constant region or a portion thereofcan be cloned, e.g., using the polymerase chain reaction and primerswhich are selected to amplify the domain of interest. To clone asequence of the immunoglobulin constant region or a portion thereof froman antibody, mRNA can be isolated from hybridoma, spleen, or lymphcells, reverse transcribed into DNA, and antibody genes amplified byPCR. PCR amplification methods are described in detail in U.S. Pat. Nos.4,683,195; 4,683,202; 4,800,159; 4,965,188; and in, e.g., “PCRProtocols: A Guide to Methods and Applications” Innis et al. eds.,Academic Press, San Diego, Calif. (1990); Ho et al. 1989. Gene 77:51;Horton et al. 1993. Methods Enzymol. 217:270). PCR may be initiated byconsensus constant region primers or by more specific primers based onthe published heavy and light chain DNA and amino acid sequences. Asdiscussed above, PCR also may be used to isolate DNA clones encoding theantibody light and heavy chains. In this case the libraries may bescreened by consensus primers or larger homologous probes, such as mouseconstant region probes. Numerous primer sets suitable for amplificationof antibody genes are known in the art (e.g., 5′ primers based on theN-terminal sequence of purified antibodies (Benhar and Pastan. 1994.Protein Engineering 7:1509); rapid amplification of cDNA ends (Ruberti,F. et al. 1994. J. Immunol. Methods 173:33); antibody leader sequences(Larrick et al. 1989 Biochem. Biophys. Res. Commun. 160:1250). Thecloning of antibody sequences is further described in Newman et al.,U.S. Pat. No. 5,658,570, filed Jan. 25, 1995, which is incorporated byreference herein.

An immunoglobulin constant region used herein can include all domainsand the hinge region or portions thereof. In one embodiment, theimmunoglobulin constant region or a portion thereof comprises CH2domain, CH3 domain, and a hinge region, i.e., an Fc region or an FcRnbinding partner.

As used herein, the term “Fc region” is defined as the portion of apolypeptide which corresponds to the Fc region of native immunoglobulin,i.e., as formed by the dimeric association of the respective Fc domainsof its two heavy chains. A native Fc region forms a homodimer withanother Fc region. In contrast, the term “genetically-fused Fc region”or “single-chain Fc region” (scFc region), as used herein, refers to asynthetic dimeric Fc region comprised of Fc domains genetically linkedwithin a single polypeptide chain (i.e., encoded in a single contiguousgenetic sequence).

In one embodiment, the “Fc region” refers to the portion of a singleimmunoglobulin heavy chain beginning in the hinge region just upstreamof the papain cleavage site (i.e. residue 216 in IgG, taking the firstresidue of heavy chain constant region to be 114) and ending at theC-terminus of the antibody. Accordingly, a complete Fc domain comprisesat least a hinge domain, a CH2 domain, and a CH3 domain.

The Fc region of an immunoglobulin constant region, depending on theimmunoglobulin isotype can include the CH2, CH3, and CH4 domains, aswell as the hinge region. Chimeric proteins comprising an Fc region ofan immunoglobulin bestow several desirable properties on a chimericprotein including increased stability, increased serum half-life (seeCapon et al., 1989, Nature 337:525) as well as binding to Fc receptorssuch as the neonatal Fc receptor (FcRn) (U.S. Pat. Nos. 6,086,875,6,485,726, 6,030,613; WO 03/077834; US2003-0235536A1), which areincorporated herein by reference in their entireties.

An immunoglobulin constant region or a portion thereof can be an FcRnbinding partner. FcRn is active in adult epithelial tissues andexpressed in the lumen of the intestines, pulmonary airways, nasalsurfaces, vaginal surfaces, colon and rectal surfaces (U.S. Pat. No.6,485,726). An FcRn binding partner is a portion of an immunoglobulinthat binds to FcRn.

The FcRn receptor has been isolated from several mammalian speciesincluding humans. The sequences of the human FcRn, monkey FcRn, ratFcRn, and mouse FcRn are known (Story et al. 1994, J. Exp. Med.180:2377). The FcRn receptor binds IgG (but not other immunoglobulinclasses such as IgA, IgM, IgD, and IgE) at relatively low pH, activelytransports the IgG transcellularly in a luminal to serosal direction,and then releases the IgG at relatively higher pH found in theinterstitial fluids. It is expressed in adult epithelial tissue (U.S.Pat. Nos. 6,485,726, 6,030,613, 6,086,875; WO 03/077834;US2003-0235536A1) including lung and intestinal epithelium (Israel etal. 1997, Immunology 92:69) renal proximal tubular epithelium (Kobayashiet al. 2002, Am. J. Physiol. Renal Physiol. 282:F358) as well as nasalepithelium, vaginal surfaces, and biliary tree surfaces.

FcRn binding partners useful in the present invention encompassmolecules that can be specifically bound by the FcRn receptor includingwhole IgG, the Fc fragment of IgG, and other fragments that include thecomplete binding region of the FcRn receptor. The region of the Fcportion of IgG that binds to the FcRn receptor has been described basedon X-ray crystallography (Burmeister et al. 1994, Nature 372:379). Themajor contact area of the Fc with the FcRn is near the junction of theCH2 and CH3 domains. Fc-FcRn contacts are all within a single Ig heavychain. The FcRn binding partners include whole IgG, the Fc fragment ofIgG, and other fragments of IgG that include the complete binding regionof FcRn. The major contact sites include amino acid residues 248,250-257, 272, 285, 288, 290-291, 308-311, and 314 of the CH2 domain andamino acid residues 385-387, 428, and 433-436 of the CH3 domain.References made to amino acid numbering of immunoglobulins orimmunoglobulin fragments, or regions, are all based on Kabat et al.1991, Sequences of Proteins of Immunological Interest, U.S. Departmentof Public Health, Bethesda, Md.

Fc regions or FcRn binding partners bound to FcRn can be effectivelyshuttled across epithelial barriers by FcRn, thus providing anon-invasive means to systemically administer a desired therapeuticmolecule. Additionally, fusion proteins comprising an Fc region or anFcRn binding partner are endocytosed by cells expressing the FcRn. Butinstead of being marked for degradation, these fusion proteins arerecycled out into circulation again, thus increasing the in vivohalf-life of these proteins. In certain embodiments, the portions ofimmunoglobulin constant regions are an Fc region or an FcRn bindingpartner that typically associates, via disulfide bonds and othernon-specific interactions, with another Fc region or another FcRnbinding partner to form dimers and higher order multimers.

Two FcRn receptors can bind a single Fc molecule. Crystallographic datasuggest that each FcRn molecule binds a single polypeptide of the Fchomodimer. In one embodiment, linking the FcRn binding partner, e.g., anFc fragment of an IgG, to a biologically active molecule provides ameans of delivering the biologically active molecule orally, buccally,sublingually, rectally, vaginally, as an aerosol administered nasally orvia a pulmonary route, or via an ocular route. In another embodiment,the chimeric protein can be administered invasively, e.g.,subcutaneously, intravenously.

An FcRn binding partner region is a molecule or a portion thereof thatcan be specifically bound by the FcRn receptor with consequent activetransport by the FcRn receptor of the Fc region. Specifically boundrefers to two molecules forming a complex that is relatively stableunder physiologic conditions. Specific binding is characterized by ahigh affinity and a low to moderate capacity as distinguished fromnonspecific binding which usually has a low affinity with a moderate tohigh capacity. Typically, binding is considered specific when theaffinity constant KA is higher than 10⁶ M⁻¹, or higher than 10⁸ M⁻¹. Ifnecessary, non-specific binding can be reduced without substantiallyaffecting specific binding by varying the binding conditions. Theappropriate binding conditions such as concentration of the molecules,ionic strength of the solution, temperature, time allowed for binding,concentration of a blocking agent (e.g. serum albumin, milk casein),etc., may be optimized by a skilled artisan using routine techniques.

In certain embodiments, a chimeric protein of the invention comprisesone or more truncated Fc regions that are nonetheless sufficient toconfer Fc receptor (FcR) binding properties to the Fc region. Forexample, the portion of an Fc region that binds to FcRn (i.e., the FcRnbinding portion) comprises from about amino acids 282-438 of IgG1, EUnumbering (with the primary contact sites being amino acids 248,250-257, 272, 285, 288, 290-291, 308-311, and 314 of the CH2 domain andamino acid residues 385-387, 428, and 433-436 of the CH3 domain. Thus,an Fc region of the invention may comprise or consist of an FcRn bindingportion. FcRn binding portions may be derived from heavy chains of anyisotype, including IgG1, IgG2, IgG3 and IgG4. In one embodiment, an FcRnbinding portion from an antibody of the human isotype IgG1 is used. Inanother embodiment, an FcRn binding portion from an antibody of thehuman isotype IgG4 is used.

In another embodiment, the “Fc region” includes an amino acid sequenceof an Fc domain or derived from an Fc domain. In certain embodiments, anFc region comprises at least one of: a hinge (e.g., upper, middle,and/or lower hinge region) domain (about amino acids 216-230 of anantibody Fc region according to EU numbering), a CH2 domain (about aminoacids 231-340 of an antibody Fc region according to EU numbering), a CH3domain (about amino acids 341-438 of an antibody Fc region according toEU numbering), a CH4 domain, or a variant, portion, or fragment thereof.In other embodiments, an Fc region comprises a complete Fc domain (i.e.,a hinge domain, a CH2 domain, and a CH3 domain). In some embodiments, anFc region comprises, consists essentially of, or consists of a hingedomain (or a portion thereof) fused to a CH3 domain (or a portionthereof), a hinge domain (or a portion thereof) fused to a CH2 domain(or a portion thereof), a CH2 domain (or a portion thereof) fused to aCH3 domain (or a portion thereof), a CH2 domain (or a portion thereof)fused to both a hinge domain (or a portion thereof) and a CH3 domain (ora portion thereof). In still other embodiments, an Fc region lacks atleast a portion of a CH2 domain (e.g., all or part of a CH2 domain). Ina particular embodiment, an Fc region comprises or consists of aminoacids corresponding to EU numbers 221 to 447.

The Fc regions denoted as F, F1, or F2 herein may be obtained from anumber of different sources. In one embodiment, an Fc region of thepolypeptide is derived from a human immunoglobulin. It is understood,however, that an Fc region may be derived from an immunoglobulin ofanother mammalian species, including for example, a rodent (e.g. amouse, rat, rabbit, guinea pig) or non-human primate (e.g. chimpanzee,macaque) species. Moreover, the polypeptide of the Fc domains orportions thereof may be derived from any immunoglobulin class, includingIgM, IgG, IgD, IgA and IgE, and any immunoglobulin isotype, includingIgGl, IgG2, IgG3 and IgG4. In another embodiment, the human isotype IgG1is used.

In certain embodiments, the Fc variant confers a change in at least oneeffector function imparted by an Fc region comprising said wild-type Fcdomain (e.g., an improvement or reduction in the ability of the Fcregion to bind to Fc receptors (e.g. FcγRI, FcγRII, or FcγRIII) orcomplement proteins (e.g. C1q), or to trigger antibody-dependentcytotoxicity (ADCC), phagocytosis, or complement-dependent cytotoxicity(CDCC)). In other embodiments, the Fc variant provides an engineeredcysteine residue.

The Fc regions of the invention may employ art-recognized Fc variantswhich are known to impart a change (e.g., an enhancement or reduction)in effector function and/or FcR or FcRn binding. Specifically, a bindingmolecule of the invention may include, for example, a change (e.g., asubstitution) at one or more of the amino acid positions disclosed inInternational PCT Publications WO88/07089A1, WO96/14339A1, WO98/05787A1,WO98/23289A1, WO99/51642A1, WO99/58572A1, WO00/09560A2, WO00/32767A1,WO00/42072A2, WO02/44215A2, WO02/060919A2, WO03/074569A2, WO04/016750A2,WO04/029207A2, WO04/035752A2, WO04/063351A2, WO04/074455A2,WO04/099249A2, WO05/040217A2, WO04/044859, WO05/070963A1, WO05/077981A2,WO05/092925A2, WO05/123780A2, WO06/019447A1, WO06/047350A2, andWO06/085967A2; US Patent Publication Nos. US2007/0231329,US2007/0231329, US2007/0237765, US2007/0237766, US2007/0237767,US2007/0243188, US20070248603, US20070286859, US20080057056; or U.S.Pat. Nos. 5,648,260; 5,739,277; 5,834,250; 5,869,046; 6,096,871;6,121,022; 6,194,551; 6,242,195; 6,277,375; 6,528,624; 6,538,124;6,737,056; 6,821,505; 6,998,253; 7,083,784; 7,404,956, and 7,317,091,each of which is incorporated by reference herein. In one embodiment,the specific change (e.g., the specific substitution of one or moreamino acids disclosed in the art) may be made at one or more of thedisclosed amino acid positions. In another embodiment, a differentchange at one or more of the disclosed amino acid positions (e.g., thedifferent substitution of one or more amino acid position disclosed inthe art) may be made.

The Fc region or FcRn binding partner of IgG can be modified accordingto well recognized procedures such as site directed mutagenesis and thelike to yield modified IgG or Fc fragments or portions thereof that willbe bound by FcRn. Such modifications include modifications remote fromthe FcRn contact sites as well as modifications within the contact sitesthat preserve or even enhance binding to the FcRn. For example, thefollowing single amino acid residues in human IgG1 Fc (Fc γ1) can besubstituted without significant loss of Fc binding affinity for FcRn:P238A, S239A, K246A, K248A, D249A, M252A, T256A, E258A, T260A, D265A,S267A, H268A, E269A, D270A, E272A, L274A, N276A, Y278A, D280A, V282A,E283A, H285A, N286A, T289A, K290A, R292A, E293A, E294A, Q295A, Y296F,N297A, S298A, Y300F, R301A, V303A, V305A, T307A, L309A, Q311A, D312A,N315A, K317A, E318A, K320A, K322A, S324A, K326A, A327Q, P329A, A330Q,P331A, E333A, K334A, T335A, S337A, K338A, K340A, Q342A, R344A, E345A,Q347A, R355A, E356A, M358A, T359A, K360A, N361A, Q362A, Y373A, S375A,D376A, A378Q, E380A, E382A, S383A, N384A, Q386A, E388A, N389A, N390A,Y391F, K392A, L398A, S400A, D401A, D413A, K414A, R416A, Q418A, Q419A,N421A, V422A, S424A, E430A, N434A, T437A, Q438A, K439A, S440A, S444A,and K447A, where for example P238A represents wild type prolinesubstituted by alanine at position number 238. As an example, a specificembodiment incorporates the N297A mutation, removing a highly conservedN-glycosylation site. In addition to alanine other amino acids may besubstituted for the wild type amino acids at the positions specifiedabove. Mutations may be introduced singly into Fc giving rise to morethan one hundred Fc regions distinct from the native Fc. Additionally,combinations of two, three, or more of these individual mutations may beintroduced together, giving rise to hundreds more Fc regions. Moreover,one of the Fc region of a construct of the invention may be mutated andthe other Fc region of the construct not mutated at all, or they bothmay be mutated but with different mutations.

Certain of the above mutations may confer new functionality upon the Fcregion or FcRn binding partner. For example, one embodiment incorporatesN297A, removing a highly conserved N-glycosylation site. The effect ofthis mutation is to reduce immunogenicity, thereby enhancing circulatinghalf-life of the Fc region, and to render the Fc region incapable ofbinding to FcγRI, FcγRIIA, FcγRIIB, and FcγRIIIA, without compromisingaffinity for FcRn (Routledge et al. 1995, Transplantation 60:847; Friendet al. 1999, Transplantation 68:1632; Shields et al. 1995, J. Biol.Chem. 276:6591). As a further example of new functionality arising frommutations described above affinity for FcRn may be increased beyond thatof wild type in some instances. This increased affinity may reflect anincreased “on” rate, a decreased “off” rate or both an increased “on”rate and a decreased “off” rate. Examples of mutations believed toimpart an increased affinity for FcRn include, but not limited to,T256A, T307A, E380A, and N434A (Shields et al. 2001, J. Biol. Chem.276:6591).

Additionally, at least three human Fc gamma receptors appear torecognize a binding site on IgG within the lower hinge region, generallyamino acids 234-237. Therefore, another example of new functionality andpotential decreased immunogenicity may arise from mutations of thisregion, as for example by replacing amino acids 233-236 of human IgG1“ELLG” to the corresponding sequence from IgG2 “PVA” (with one aminoacid deletion). It has been shown that FcγR1, FcγRII, and FcγRIII, whichmediate various effector functions will not bind to IgG1 when suchmutations have been introduced. Ward and Ghetie 1995, TherapeuticImmunology 2:77 and Armour et al. 1999, Eur. J. Immunol. 29:2613.

In one embodiment, the immunoglobulin constant region or a portionthereof, e.g, an Fc region, is a polypeptide including the sequencePKNSSMISNTP (SEQ ID NO: 3) and optionally further including a sequenceselected from HQSLGTQ (SEQ ID NO: 4), HQNLSDGK (SEQ ID NO: 5), HQNISDGK(SEQ ID NO: 6), or VISSHLGQ (SEQ ID NO: 7) (U.S. Pat. No. 5,739,277).

In another embodiment, the immunoglobulin constant region or a portionthereof comprises an amino acid sequence in the hinge region or aportion thereof that forms one or more disulfide bonds with anotherimmunoglobulin constant region or a portion thereof. The disulfide bondby the immunoglobulin constant region or a portion thereof places thefirst polypeptide comprising FVIII and the second polypeptide comprisingthe VWF fragment together so that endogenous VWF does not replace theVWF fragment and does not bind to the FVIII. Therefore, the disulfidebond between the first immunoglobulin constant region or a portionthereof and a second immunoglobulin constant region or a portion thereofprevents interaction between endogenous VWF and the FVIII protein. Thisinhibition of interaction between the VWF and the FVIII protein allowsthe half-life of the FVIII protein to go beyond the two fold limit. Thehinge region or a portion thereof can further be linked to one or moredomains of CH1, CH2, CH3, a fragment thereof, and any combinationsthereof. In a particular example, an immunoglobulin constant region or aportion thereof comprises a hinge region and CH2 region (e.g., aminoacids 221-340 of an Fc region).

In certain embodiments, the immunoglobulin constant region or a portionthereof is hemi-glycosylated. For example, the chimeric proteincomprising two Fc regions or FcRn binding partners may contain a first,glycosylated, Fc region (e.g., a glycosylated CH2 region) or FcRnbinding partner and a second, aglycosylated, Fc region (e.g., anaglycosylated CH2 region) or FcRn binding partner. In one embodiment, alinker may be interposed between the glycosylated and aglycosylated Fcregions. In another embodiment, the Fc region or FcRn binding partner isfully glycosylated, i.e., all of the Fc regions are glycosylated. Inother embodiments, the Fc region may be aglycosylated, i.e., none of theFc moieties are glycosylated.

In certain embodiments, a chimeric protein of the invention comprises anamino acid substitution to an immunoglobulin constant region or aportion thereof (e.g., Fc variants), which alters theantigen-independent effector functions of the Ig constant region, inparticular the circulating half-life of the protein.

Such proteins exhibit either increased or decreased binding to FcRn whencompared to proteins lacking these substitutions and, therefore, have anincreased or decreased half-life in serum, respectively. Fc variantswith improved affinity for FcRn are anticipated to have longer serumhalf-lives, and such molecules have useful applications in methods oftreating mammals where long half-life of the administered polypeptide isdesired, e.g., to treat a chronic disease or disorder (see, e.g, U.S.Pat. Nos. 7,348,004, 7,404,956, and 7,862,820). In contrast, Fc variantswith decreased FcRn binding affinity are expected to have shorterhalf-lives, and such molecules are also useful, for example, foradministration to a mammal where a shortened circulation time may beadvantageous, e.g. for in vivo diagnostic imaging or in situations wherethe starting polypeptide has toxic side effects when present in thecirculation for prolonged periods. Fc variants with decreased FcRnbinding affinity are also less likely to cross the placenta and, thus,are also useful in the treatment of diseases or disorders in pregnantwomen. In addition, other applications in which reduced FcRn bindingaffinity may be desired include those applications in which localizationthe brain, kidney, and/or liver is desired. In one exemplary embodiment,the chimeric protein of the invention exhibit reduced transport acrossthe epithelium of kidney glomeruli from the vasculature. In anotherembodiment, the chimeric protein of the invention exhibit reducedtransport across the blood brain barrier (BBB) from the brain, into thevascular space. In one embodiment, a protein with altered FcRn bindingcomprises at least one Fc region or FcRn binding partner (e.g, one ortwo Fc regions or FcRn binding partners) having one or more amino acidsubstitutions within the “FcRn binding loop” of an Ig constant region.The FcRn binding loop is comprised of amino acid residues 280-299(according to EU numbering) of a wild-type, full-length, Fc region. Inother embodiments, an Ig constant region or a portion thereof in achimeric protein of the invention having altered FcRn binding affinitycomprises at least one Fc region or FcRn binding partner having one ormore amino acid substitutions within the 15 {acute over (Å)} FcRn“contact zone.” As used herein, the term 15 {acute over (Å)} FcRn“contact zone” includes residues at the following positions of awild-type, full-length Fc moiety: 243-261, 275-280, 282-293, 302-319,336-348, 367, 369, 372-389, 391, 393, 408, 424, 425-440 (EU numbering).In other embodiments, a Ig constant region or a portion thereof of theinvention having altered FcRn binding affinity comprises at least one Fcregion or FcRn binding partner having one or more amino acidsubstitutions at an amino acid position corresponding to any one of thefollowing EU positions: 256, 277-281, 283-288, 303-309, 313, 338, 342,376, 381, 384, 385, 387, 434 (e.g., N434A or N434K), and 438. Exemplaryamino acid substitutions which altered FcRn binding activity aredisclosed in International PCT Publication No. WO05/047327 which isincorporated by reference herein.

An Fc region or FcRn binding partner used in the invention may alsocomprise an art recognized amino acid substitution which alters theglycosylation of the chimeric protein. For example, the Fc region orFcRn binding partner of the chimeric protein linked to a VWF fragment ora FVIII protein may comprise an Fc region having a mutation leading toreduced glycosylation (e.g., N- or O-linked glycosylation) or maycomprise an altered glycoform of the wild-type Fc moiety (e.g., a lowfucose or fucose-free glycan).

In one embodiment, an unprocessed chimeric protein of the invention maycomprise a genetically fused Fc region (i.e., scFc region) having two ormore of its constituent Ig constant region or a portion thereofindependently selected from the Ig constant region or a portion thereofdescribed herein. In one embodiment, the Fc regions of a dimeric Fcregion are the same. In another embodiment, at least two of the Fcregions are different. For example, the Fc regions or FcRn bindingpartners of the proteins of the invention comprise the same number ofamino acid residues or they may differ in length by one or more aminoacid residues (e.g., by about 5 amino acid residues (e.g., 1, 2, 3, 4,or 5 amino acid residues), about 10 residues, about 15 residues, about20 residues, about 30 residues, about 40 residues, or about 50residues). In yet other embodiments, the Fc regions or FcRn bindingpartners of the protein of the invention may differ in sequence at oneor more amino acid positions. For example, at least two of the Fcregions or FcRn binding partners may differ at about 5 amino acidpositions (e.g., 1, 2, 3, 4, or 5 amino acid positions), about 10positions, about 15 positions, about 20 positions, about 30 positions,about 40 positions, or about 50 positions).

2) Albumin or Fragment, or Variant Thereof

In certain embodiments, the heterologous moiety linked to the VWFfragment or linked to a FVIII protein is albumin or a functionalfragment thereof. In other embodiments, a chimeric protein of theinvention comprises a FVIII protein and albumin or a fragment thereof,wherein the albumin or a fragment thereof shields or protects the VWFbinding site on the FVIII protein, thereby inhibiting or preventinginteraction of the FVIII protein with endogenous VWF.

Human serum albumin (HSA, or HA), a protein of 609 amino acids in itsfull-length form, is responsible for a significant proportion of theosmotic pressure of serum and also functions as a carrier of endogenousand exogenous ligands. The term “albumin” as used herein includesfull-length albumin or a functional fragment, variant, derivative, oranalog thereof.

In one embodiment, the chimeric protein comprises the VWF fragmentdescribed herein and albumin, fragment, or variant thereof, wherein theVWF fragment is linked to albumin or a fragment or variant thereof. Inanother embodiment, the chimeric protein comprises the VWF fragment anda FVIII protein, which are bound to each other, wherein the VWF fragmentis linked to albumin or a fragment or variant thereof, the proteinhaving VIII activity is linked to albumin or a fragment or variantthereof, or both the VWF fragment and the protein having VIII activityare linked to albumin or a fragment or variant thereof. In otherembodiments, the chimeric protein comprises the VWF fragment linked toalbumin or a fragment or variant thereof is further linked to aheterologous moiety selected from the group consisting of animmunoglobulin constant region or a portion thereof (e.g., an Fcregion), a PAS sequence, HES, and PEG. In still other embodiments, thechimeric protein comprises the VWF fragment and a FVIII protein, whichare bound to each other, wherein the FVIII protein is linked to albuminor a fragment or variant thereof and further linked to a heterologousmoiety selected from the group consisting of an immunoglobulin constantregion or a portion thereof (e.g., an Fc region), a PAS sequence, HES,and PEG. In yet other embodiments, the chimeric protein comprises theVWF fragment linked to albumin or a fragment or variant thereof and aFVIII protein linked to albumin or a fragment or variant thereof, whichare bound to each other, wherein the VWF fragment activity is furtherlinked to a first heterologous moiety selected from the group consistingof an immunoglobulin constant region or a portion thereof (e.g., an Fcregion), a PAS sequence, HES, and PEG and wherein the FVIII proteinactivity is further linked to a second heterologous moiety selected fromthe group consisting of an immunoglobulin constant region or a portionthereof (e.g., an Fc region), a PAS sequence, HES, and PEG.

In other embodiments, the heterologous moiety linked to the VWF fragmentor the FVIII protein is albumin or a fragment or variant thereof, whichextends (or is capable of extending) the half-life of the VWF fragmentor the FVIII protein. Further examples of albumin or the fragments orvariants thereof are disclosed in US Pat. Publ. Nos. 2008/0194481A1,2008/0004206 A1, 2008/0161243 A1, 2008/0261877 A1, or 2008/0153751 A1 orPCT Appl. Publ. Nos. 2008/033413 A2, 2009/058322 A1, or 2007/021494 A2.

3) Albumin Binding Moiety

In certain embodiments, the heterologous moiety linked to the VWFfragment or the FVIII protein is an albumin binding moiety, whichcomprises an albumin binding peptide, a bacterial albumin bindingdomain, an albumin-binding antibody fragment, or any combinationsthereof. For example, the albumin binding protein can be a bacterialalbumin binding protein, an antibody or an antibody fragment includingdomain antibodies (see U.S. Pat. No. 6,696,245). An albumin bindingprotein, for example, can be a bacterial albumin binding domain, such asthe one of streptococcal protein G (Konig, T. and Skerra, A. (1998) J.Immunol. Methods 218, 73-83). Other examples of albumin binding peptidesthat can be used as conjugation partner are, for instance, those havinga Cys-Xaa₁-Xaa₂-Xaa₃-Xaa₄-Cys consensus sequence, wherein Xaa₁ is Asp,Asn, Ser, Thr, or Trp; Xaa₂ is Asn, Gln, H is, Ile, Leu, or Lys; Xaa₃ isAla, Asp, Phe, Trp, or Tyr; and Xaa₄ is Asp, Gly, Leu, Phe, Ser, or Thras described in US patent application 2003/0069395 or Dennis et al.(Dennis et al. (2002) J. Biol. Chem. 277, 35035-35043).

4) PAS Sequence

In other embodiments, the heterologous moiety linked to the VWF fragmentor to the FVIII protein is a PAS sequence. In one embodiment, thechimeric protein comprises a VWF fragment described herein and a PASsequence, wherein the VWF fragment is linked to the PAS sequence. Inanother embodiment, a chimeric protein of the invention comprises aFVIII protein and a PAS sequence, wherein the PAS sequence shields orprotects the VWF binding site on the FVIII protein, thereby inhibitingor preventing interaction of the FVIII protein with endogenous VWF.

A PAS sequence, as used herein, means an amino acid sequence comprisingmainly alanine and serine residues or comprising mainly alanine, serine,and proline residues, the amino acid sequence forming random coilconformation under physiological conditions. Accordingly, the PASsequence is a building block, an amino acid polymer, or a sequencecassette comprising, consisting essentially of, or consisting ofalanine, serine, and proline which can be used as a part of theheterologous moiety in the chimeric protein. Yet, the skilled person isaware that an amino acid polymer also may form random coil conformationwhen residues other than alanine, serine, and proline are added as aminor constituent in the PAS sequence. The term “minor constituent” asused herein means that amino acids other than alanine, serine, andproline may be added in the PAS sequence to a certain degree, e.g., upto about 12%, i.e., about 12 of 100 amino acids of the PAS sequence, upto about 10%, i.e. about 10 of 100 amino acids of the PAS sequence, upto about 9%, i.e., about 9 of 100 amino acids, up to about 8%, i.e.,about 8 of 100 amino acids, about 6%, i.e., about 6 of 100 amino acids,about 5%, i.e., about 5 of 100 amino acids, about 4%, i.e., about 4 of100 amino acids, about 3%, i.e., about 3 of 100 amino acids, about 2%,i.e., about 2 of 100 amino acids, about 1%, i.e., about 1 of 100 of theamino acids. The amino acids different from alanine, serine and prolinemay be selected from the group consisting of Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Tyr, and Val.

Under physiological conditions, the PAS sequence stretch forms a randomcoil conformation and thereby can mediate an increased in vivo and/or invitro stability to the VWF factor or the protein of coagulationactivity. Since the random coil domain does not adopt a stable structureor function by itself, the biological activity mediated by the VWFfragment or the FVIII protein to which it is fused is essentiallypreserved. In other embodiments, the PAS sequences that form random coildomain are biologically inert, especially with respect to proteolysis inblood plasma, immunogenicity, isoelectric point/electrostatic behavior,binding to cell surface receptors or internalization, but are stillbiodegradable, which provides clear advantages over synthetic polymerssuch as PEG.

Non-limiting examples of the PAS sequences forming random coilconformation comprise an amino acid sequence selected from the groupconsisting of ASPAAPAPASPAAPAPSAPA (SEQ ID NO: 8), AAPASPAPAAPSAPAPAAPS(SEQ ID NO: 9), APSSPSPSAPSSPSPASPSS (SEQ ID NO: 10),APSSPSPSAPSSPSPASPS (SEQ ID NO: 11), SSPSAPSPSSPASPSPSSPA (SEQ ID NO:12), AASPAAPSAPPAAASPAAPSAPPA (SEQ ID NO: 13) and ASAAAPAAASAAASAPSAAA(SEQ ID NO: 14) or any combinations thereof. Additional examples of PASsequences are known from, e.g., US Pat. Publ. No. 2010/0292130 A1 andPCT Appl. Publ. No. WO 2008/155134 A1.

5) HAP Sequence

In certain embodiments, the heterologous moiety linked to the VWFfragment or the FVIII protein is a glycine-rich homo-amino-acid polymer(HAP). The HAP sequence can comprise a repetitive sequence of glycine,which has at least 50 amino acids, at least 100 amino acids, 120 aminoacids, 140 amino acids, 160 amino acids, 180 amino acids, 200 aminoacids, 250 amino acids, 300 amino acids, 350 amino acids, 400 aminoacids, 450 amino acids, or 500 amino acids in length. In one embodiment,the HAP sequence is capable of extending half-life of a moiety fused toor linked to the HAP sequence. Non-limiting examples of the HAP sequenceincludes, but are not limited to (Gly)_(n), (Gly₄Ser)_(n) orS(Gly₄Ser)_(n), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20. In one embodiment, n is 20, 21, 22, 23,24, 25, 26, 26, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40.In another embodiment, n is 50, 60, 70, 80, 90, 100, 110, 120, 130, 140,150, 160, 170, 180, 190, or 200. See, e.g., Schlapschy M et al., ProteinEng. Design Selection, 20: 273-284 (2007).

6) Transferrin or Fragment Thereof

In certain embodiments, the heterologous moiety linked to the VWFfragment or the FVIII protein is transferrin or a fragment thereof. Anytransferrin may be used to make the chimeric proteins of the invention.As an example, wild-type human Tf (Tf) is a 679 amino acid protein, ofapproximately 75 KDa (not accounting for glycosylation), with two maindomains, N (about 330 amino acids) and C (about 340 amino acids), whichappear to originate from a gene duplication. See GenBank accessionnumbers NM001063, XM002793, M12530, XM039845, XM 039847 and 595936(www.ncbi.nlm.nih.gov/), all of which are herein incorporated byreference in their entirety. Transferrin comprises two domains, N domainand C domain. N domain comprises two subdomains, N1 domain and N2domain, and C domain comprises two subdomains, C1 domain and C2 domain.

In one embodiment, the transferrin portion of the chimeric proteinincludes a transferrin splice variant. In one example, a transferrinsplice variant can be a splice variant of human transferrin, e.g.,Genbank Accession AAA61140. In another embodiment, the transferrinportion of the chimeric protein includes one or more domains of thetransferrin sequence, e.g., N domain, C domain, N1 domain, N2 domain, C1domain, C2 domain or any combinations thereof.

7) Polymer, e.g., Polyethylene Glycol (PEG)

In other embodiments, the heterologous moiety attached to the VWFfragment or the protein having clotting activity, e.g. FVIII activity,is a soluble polymer known in the art, including, but not limited to,polyethylene glycol, ethylene glycol/propylene glycol copolymers,carboxymethylcellulose, dextran, or polyvinyl alcohol. The heterologousmoiety such as soluble polymer can be attached to any positions withinthe VWF fragment or the FVIII protein or the N- or C-terminus. In stillother embodiments, a chimeric protein of the invention comprises a FVIIIprotein and PEG, wherein PEG shields or protects the VWF binding site onthe FVIII protein, thereby inhibiting or preventing interaction of theFVIII protein with endogenous VWF.

In certain embodiments, the chimeric protein comprises the VWF fragmentdescribed herein and PEG, wherein the VWF fragment is linked to PEG. Inanother embodiment, the chimeric protein comprises the VWF fragment anda FVIII protein, which are bound to each other, wherein the VWF fragmentis linked to PEG, the FVIII protein is linked to PEG, or both the VWFfragment and the FVIII protein are linked to PEG. In other embodiments,the chimeric protein comprising the VWF fragment linked to PEG isfurther linked to a heterologous moiety selected from the groupconsisting of an immunoglobulin constant region or a portion thereof(e.g., an Fc region), a PAS sequence, HES, and albumin, fragment, orvariant thereof. In still other embodiments, the chimeric proteincomprises the VWF fragment and a FVIII protein, which are bound to eachother, wherein the FVIII protein is further linked to a heterologousmoiety selected from the group consisting of an immunoglobulin constantregion or a portion thereof (e.g., an Fc region), a PAS sequence, HES,and albumin, fragment, or variant thereof. In yet other embodiments, thechimeric protein comprises the VWF fragment linked to PEG and a FVIIIprotein linked to PEG, which are bound to each other, wherein the VWFfragment activity is further linked to a first heterologous moietyselected from the group consisting of an immunoglobulin constant regionor a portion thereof (e.g., an Fc region), a PAS sequence, HES, andalbumin, fragment, or variant thereof and wherein the FVIII proteinactivity is further linked to a second heterologous moiety selected fromthe group consisting of an immunoglobulin constant region or a portionthereof (e.g., an Fc region), a PAS sequence, HES, and albumin,fragment, or variant thereof.

Also provided by the invention are chemically modified derivatives ofthe chimeric protein of the invention which may provide additionaladvantages such as increased solubility, stability and circulating timeof the polypeptide, or decreased immunogenicity (see U.S. Pat. No.4,179,337). The chemical moieties for modification can be selected fromthe group consisting of water soluble polymers including, but notlimited to, polyethylene glycol, ethylene glycol/propylene glycolcopolymers, carboxymethylcellulose, dextran, and polyvinyl alcohol. Thechimeric protein may be modified at random positions within the moleculeor at the N- or C-terminus, or at predetermined positions within themolecule and may include one, two, three or more attached chemicalmoieties.

The polymer can be of any molecular weight, and can be branched orunbranched. For polyethylene glycol, in one embodiment, the molecularweight is between about 1 kDa and about 100 kDa for ease in handling andmanufacturing. Other sizes may be used, depending on the desired profile(e.g., the duration of sustained release desired, the effects, if any onbiological activity, the ease in handling, the degree or lack ofantigenicity and other known effects of the polyethylene glycol to aprotein or analog). For example, the polyethylene glycol may have anaverage molecular weight of about 200, 500, 1000, 1500, 2000, 2500,3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500,9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000,13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500,18,000, 18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000, 40,000,45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000,90,000, 95,000, or 100,000 kDa.

In some embodiments, the polyethylene glycol may have a branchedstructure. Branched polyethylene glycols are described, for example, inU.S. Pat. No. 5,643,575; Morpurgo et al., Appl. Biochem. Biotechnol.56:59-72 (1996); Vorobjev et al., Nucleosides Nucleotides 18:2745-2750(1999); and Caliceti et al., Bioconjug. Chem. 10:638-646 (1999), each ofwhich is incorporated herein by reference in its entirety.

The number of polyethylene glycol moieties attached to each chimericprotein, the VWF fragment, or the FVIII protein of the invention (i.e.,the degree of substitution) may also vary. For example, the pegylatedproteins of the invention may be linked, on average, to 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 12, 15, 17, 20, or more polyethylene glycol molecules.Similarly, the average degree of substitution within ranges such as 1-3,2-4, 3-5, 4-6, 5-7, 6-8, 7-9, 8-10, 9-11, 10-12, 11-13, 12-14, 13-15,14-16, 15-17, 16-18, 17-19, or 18-20 polyethylene glycol moieties perprotein molecule. Methods for determining the degree of substitution arediscussed, for example, in Delgado et al., Crit. Rev. Thera. DrugCarrier Sys. 9:249-304 (1992).

In some embodiments, the FVIII protein may be PEGylated. PEGylatedFactor VIII can refer to a conjugate formed between Factor VIII and atleast one polyethylene glycol (PEG) molecule.

In other embodiments, a FVIII protein used in the invention isconjugated to one or more polymers. The polymer can be water-soluble andcovalently or non-covalently attached to Factor VIII or other moietiesconjugated to Factor VIII. Non-limiting examples of the polymer can bepoly(alkylene oxide), poly(vinyl pyrrolidone), poly(vinyl alcohol),polyoxazoline, or poly(acryloylmorpholine). Additional types ofpolymer-conjugated FVIII are disclosed in U.S. Pat. No. 7,199,223.

8) Hydroxyethyl Starch (HES)

In certain embodiments, the heterologous moiety linked to the VWFfragment or the FVIII protein is a polymer, e.g., hydroxyethyl starch(HES) or a derivative thereof. In one embodiment, a chimeric proteincomprises a VWF fragment described herein and HES, wherein the VWFfragment is linked to HES. In other embodiments, a chimeric protein ofthe invention comprises a FVIII protein fused to hydroxyethyl starch(HES), wherein the hydroxyethyl starch or a derivative thereof shieldsor protects the VWF binding site on the FVIII protein from endogenousVWF, thereby inhibiting or preventing interaction of the FVIII proteinwith endogenous VWF.

Hydroxyethyl starch (HES) is a derivative of naturally occurringamylopectin and is degraded by alpha-amylase in the body. HES is asubstituted derivative of the carbohydrate polymer amylopectin, which ispresent in corn starch at a concentration of up to 95% by weight. HESexhibits advantageous biological properties and is used as a bloodvolume replacement agent and in hemodilution therapy in the clinics(Sommermeyer et al., Krankenhauspharmazie, 8(8), 271-278 (1987); andWeidler et al., Arzneim.-Forschung/Drug Res., 41, 494-498 (1991)).

Amylopectin contains glucose moieties, wherein in the main chainalpha-1,4-glycosidic bonds are present and at the branching sitesalpha-1,6-glycosidic bonds are found. The physical-chemical propertiesof this molecule are mainly determined by the type of glycosidic bonds.Due to the nicked alpha-1,4-glycosidic bond, helical structures withabout six glucose-monomers per turn are produced. The physico-chemicalas well as the biochemical properties of the polymer can be modified viasubstitution. The introduction of a hydroxyethyl group can be achievedvia alkaline hydroxyethylation. By adapting the reaction conditions itis possible to exploit the different reactivity of the respectivehydroxy group in the unsubstituted glucose monomer with respect to ahydroxyethylation. Owing to this fact, the skilled person is able toinfluence the substitution pattern to a limited extent.

HES is mainly characterized by the molecular weight distribution and thedegree of substitution. The degree of substitution, denoted as DS,relates to the molar substitution, is known to the skilled people. SeeSommermeyer et al., Krankenhauspharmazie, 8(8), 271-278 (1987), as citedabove, in particular p. 273.

In one embodiment, hydroxyethyl starch has a mean molecular weight(weight mean) of from 1 to 300 kD, from 2 to 200 kD, from 3 to 100 kD,or from 4 to 70 kD. hydroxyethyl starch can further exhibit a molardegree of substitution of from 0.1 to 3, preferably 0.1 to 2, morepreferred, 0.1 to 0.9, preferably 0.1 to 0.8, and a ratio between C2:C6substitution in the range of from 2 to 20 with respect to thehydroxyethyl groups. A non-limiting example of HES having a meanmolecular weight of about 130 kD is a HES with a degree of substitutionof 0.2 to 0.8 such as 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, or 0.8, preferablyof 0.4 to 0.7 such as 0.4, 0.5, 0.6, or 0.7. In a specific embodiment,HES with a mean molecular weight of about 130 kD is VOLUVEN® fromFresenius. VOLUVEN® is an artificial colloid, employed, e.g., for volumereplacement used in the therapeutic indication for therapy andprophylaxis of hypovolaemia. The characteristics of VOLUVEN® are a meanmolecular weight of 130,000+/−20,000 D, a molar substitution of 0.4 anda C2:C6 ratio of about 9:1. In other embodiments, ranges of the meanmolecular weight of hydroxyethyl starch are, e.g., 4 to 70 kD or 10 to70 kD or 12 to 70 kD or 18 to 70 kD or 50 to 70 kD or 4 to 50 kD or 10to 50 kD or 12 to 50 kD or 18 to 50 kD or 4 to 18 kD or 10 to 18 kD or12 to 18 kD or 4 to 12 kD or 10 to 12 kD or 4 to 10 kD. In still otherembodiments, the mean molecular weight of hydroxyethyl starch employedis in the range of from more than 4 kD and below 70 kD, such as about 10kD, or in the range of from 9 to 10 kD or from 10 to 11 kD or from 9 to11 kD, or about 12 kD, or in the range of from 11 to 12 kD) or from 12to 13 kD or from 11 to 13 kD, or about 18 kD, or in the range of from 17to 18 kD or from 18 to 19 kD or from 17 to 19 kD, or about 30 kD, or inthe range of from 29 to 30, or from 30 to 31 kD, or about 50 kD, or inthe range of from 49 to 50 kD or from 50 to 51 kD or from 49 to 51 kD.

In certain embodiments, the heterologous moiety can be mixtures ofhydroxyethyl starches having different mean molecular weights and/ordifferent degrees of substitution and/or different ratios of C2:C6substitution. Therefore, mixtures of hydroxyethyl starches may beemployed having different mean molecular weights and different degreesof substitution and different ratios of C2:C6 substitution, or havingdifferent mean molecular weights and different degrees of substitutionand the same or about the same ratio of C2:C6 substitution, or havingdifferent mean molecular weights and the same or about the same degreeof substitution and different ratios of C2:C6 substitution, or havingthe same or about the same mean molecular weight and different degreesof substitution and different ratios of C2:C6 substitution, or havingdifferent mean molecular weights and the same or about the same degreeof substitution and the same or about the same ratio of C2:C6substitution, or having the same or about the same mean molecularweights and different degrees of substitution and the same or about thesame ratio of C2:C6 substitution, or having the same or about the samemean molecular weight and the same or about the same degree ofsubstitution and different ratios of C2:C6 substitution, or having aboutthe same mean molecular weight and about the same degree of substitutionand about the same ratio of C2:C6 substitution.

9) Polysialic Acids (PSA)

In certain embodiments, the non-polypeptide heterologous moiety linkedto the VWF fragment or the FVIII protein is a polymer, e.g., polysialicacids (PSAs) or a derivative thereof. Polysialic acids (PSAs) arenaturally occurring unbranched polymers of sialic acid produced bycertain bacterial strains and in mammals in certain cells Roth J., etal. (1993) in Polysialic Acid: From Microbes to Man, eds Roth J.,Rutishauser U., Troy F. A. (Birkhauser Verlag, Basel, Switzerland), pp335-348. They can be produced in various degrees of polymerization fromn=about 80 or more sialic acid residues down to n=2 by limited acidhydrolysis or by digestion with neuraminidases, or by fractionation ofthe natural, bacterially derived forms of the polymer. The compositionof different polysialic acids also varies such that there arehomopolymeric forms i.e. the alpha-2,8-linked polysialic acid comprisingthe capsular polysaccharide of E. coli strain K1 and the group-Bmeningococci, which is also found on the embryonic form of the neuronalcell adhesion molecule (N-CAM). Heteropolymeric forms also exist—such asthe alternating alpha-2,8 alpha-2,9 polysialic acid of E. coli strainK92 and group C polysaccharides of N. meningitidis. Sialic acid may alsobe found in alternating copolymers with monomers other than sialic acidsuch as group W135 or group Y of N. meningitidis. Polysialic acids haveimportant biological functions including the evasion of the immune andcomplement systems by pathogenic bacteria and the regulation of glialadhesiveness of immature neurons during foetal development (wherein thepolymer has an anti-adhesive function) Cho and Troy, P.N.A.S., USA, 91(1994) 11427-11431, although there are no known receptors for polysialicacids in mammals. The alpha-2,8-linked polysialic acid of E. coli strainK1 is also known as ‘colominic acid’ and is used (in various lengths) toexemplify the present invention. Various methods of attaching orconjugating polysialic acids to a polypeptide have been described (forexample, see U.S. Pat. No. 5,846,951; WO-A-0187922, and US 2007/0191597A1, which are incorporated herein by reference in their entireties.

C) FVIII Protein

“A FVIII protein” as used herein means a functional FVIII polypeptide inits normal role in coagulation, unless otherwise specified. The term aFVIII protein includes a functional fragment, variant, analog, orderivative thereof that retains the function of full-length wild-typeFactor VIII in the coagulation pathway. “A FVIII protein” is usedinterchangeably with FVIII polypeptide (or protein) or FVIII. Examplesof the FVIII functions include, but not limited to, an ability toactivate coagulation, an ability to act as a cofactor for factor IX, oran ability to form a tenase complex with factor IX in the presence ofCa2+ and phospholipids, which then converts Factor X to the activatedform Xa. The FVIII protein can be the human, porcine, canine, rat, ormurine FVIII protein. In addition, comparisons between FVIII from humansand other species have identified conserved residues that are likely tobe required for function (Cameron et al., Thromb. Haemost. 79:317-22(1998); U.S. Pat. No. 6,251,632).

A number of tests are available to assess the function of thecoagulation system: activated partial thromboplastin time (aPTT) test,chromogenic assay, ROTEM assay, prothrombin time (PT) test (also used todetermine INR), fibrinogen testing (often by the Clauss method),platelet count, platelet function testing (often by PFA-100), TCT,bleeding time, mixing test (whether an abnormality corrects if thepatient's plasma is mixed with normal plasma), coagulation factorassays, antiphosholipid antibodies, D-dimer, genetic tests (e.g. factorV Leiden, prothrombin mutation G20210A), dilute Russell's viper venomtime (dRVVT), miscellaneous platelet function tests, thromboelastography(TEG or Sonoclot), thromboelastometry (TEM®, e.g, ROTEM®), or euglobulinlysis time (ELT).

The aPTT test is a performance indicator measuring the efficacy of boththe “intrinsic” (also referred to the contact activation pathway) andthe common coagulation pathways. This test is commonly used to measureclotting activity of commercially available recombinant clottingfactors, e.g., FVIII or FIX. It is used in conjunction with prothrombintime (PT), which measures the extrinsic pathway.

ROTEM analysis provides information on the whole kinetics ofhaemostasis: clotting time, clot formation, clot stability and lysis.The different parameters in thromboelastometry are dependent on theactivity of the plasmatic coagulation system, platelet function,fibrinolysis, or many factors which influence these interactions. Thisassay can provide a complete view of secondary haemostasis.

The FVIII polypeptide and polynucleotide sequences are known, as aremany functional fragments, mutants and modified versions. Examples ofhuman FVIII sequences (full-length) are shown as subsequences in SEQ IDNO: 16 or 18.

TABLE 2Full-length FVIII (FVIII signal peptide underlined; FVIII heavy chain is doubleunderlined; B domain is italicized; and FVIII light chain is in plain text)Signal Peptide: (SEQ ID NO: 15) MQIELSTCFFLCLLRFCFS Mature Factor VIII(SEQ ID NO: 16)*

LLRQSRTPHGLSLSDLQEAKYETPSDDPSPGAIDSNNSLSEMTHPPPQLHHSGDMVPTPESGLQLRLNEYLGTTAATELKKLD 

KVSSTSNNLISTI 

SDNLAAGTDNTSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSESGRL 

KGKRAHGPALLTKDNAL 

KVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEPKKVTPLIHDRMLMDKNATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPESARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGNFLSEKNKVVVGKGEFTKDVGLKEMVFPSSRNLFLTNLDNL 

ENNTHNQEKKIQEEIEKKETLIQENVVLPQIHTVTGTKNFNKNLPLLSTPQNVEGSYDGAYAPVLQDFPSLNDSTNRTKKHTAHFSKKGEEENLEGLGHQTKQIVEKYACTTRLSPNTSQQNPVTQPSKRALKQFRLPLEETELEKRIIVDDTSTQWSK 

MKHLTPSTLTQIDY 

EKE 

GAITQS RLSDCLTRSHSIPQANRSPLPIAKVSSFPSIRPIYLTRVL 

QDNSSHLPAASYRKKDSGVQESSHPLQGAKKNNLSLAILTLEMTGDQREVGSLGTSATNSVTYKKVENTVLPKPDLPKTSG 

VELLPKVHIYQKDLFPTETSNGSPGHLDLVEGSLLQGTEGALKWNEANRPGKVPPLRVATESSAKTPSKLLDPLAWDNHYGTQIPKEEWKSQEKSPEKTAPKKKDTILSLNACESNHAIAAIN 

GQNKPEIEVTWAKQG 

TERLCSQNPPVLKR 

Q 

EITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY

indicates data missing or illegible when filed

TABLE 3 Nucleotide Sequence Encoding Full-Length FVIII (SEQ ID NO: 17)*661                                         ATG CAAATAGAGC TCTCCACCTG721 CTTCTTTCTG TGCCTTTTGC GATTCTGCTT TAGTGCCACC AGAAGATACT ACCTGGGTGC781 AGTGGAACTG TCATGGGACT ATATGCAAAG TGATCTCGGT GAGCTGCCTG TGGACGCAAG841 ATTTCCTCCT AGAGTGCCAA AATCTTTTCC ATTCAACACC TCAGTCGTGT ACAAAAAGAC901 TCTGTTTGTA GAATTCACGG ATCACCTTTT CAACATCGCT AAGCCAAGGC CACCCTGGAT961 GGGTCTGCTA GGTCCTACCA TCCAGGCTGA GGTTTATGAT ACAGTGGTCA TTACACTTAA1021 GAACATGGCT TCCCATCCTG TCAGTCTTCA TGCTGTTGGT GTATCCTACT GGAAAGCTTC1081 TGAGGGAGCT GAATATGATG ATCAGACCAG TCAAAGGGAG AAAGAAGATG ATAAAGTCTT1141 CCCTGGTGGA AGCCATACAT ATGTCTGGCA GGTCCTGAAA GAGAATGGTC CAATGGCCTC1201 TGACCCACTG TGCCTTACCT ACTCATATCT TTCTCATGTG GACCTGGTAA AAGACTTGAA1261 TTCAGGCCTC ATTGGAGCCC TACTAGTATG TAGAGAAGGG AGTCTGGCCA AGGAAAAGAC1321 ACAGACCTTG CACAAATTTA TACTACTTTT TGCTGTATTT GATGAAGGGA AAAGTTGGCA1381 CTCAGAAACA AAGAACTCCT TGATGCAGGA TAGGGATGCT GCATCTGCTC GGGCCTGGCC1441 TAAAATGCAC ACAGTCAATG GTTATGTAAA CAGGTCTCTG CCAGGTCTGA TTGGATGCCA1501 CAGGAAATCA GTCTATTGGC ATGTGATTGG AATGGGCACC ACTCCTGAAG TGCACTCAAT1561 ATTCCTCGAA GGTCACACAT TTCTTGTGAG GAACCATCGC CAGGCGTCCT TGGAAATCTC1621 GCCAATAACT TTCCTTACTG CTCAAACACT CTTGATGGAC CTTGGACAGT TTCTACTGTT1681 TTGTCATATC TCTTCCCACC AACATGATGG CATGGAAGCT TATGTCAAAG TAGACAGCTG1741 TCCAGAGGAA CCCCAACTAC GAATGAAAAA TAATGAAGAA GCGGAAGACT ATGATGATGA1801 TCTTACTGAT TCTGAAATGG ATGTGGTCAG GTTTGATGAT GACAACTCTC CTTCCTTTAT1861 CCAAATTCGC TCAGTTGCCA AGAAGCATCC TAAAACTTGG GTACATTACA TTGCTGCTGA1921 AGAGGAGGAC TGGGACTATG CTCCCTTAGT CCTCGCCCCC GATGACAGAA GTTATAAAAG1981 TCAATATTTG AACAATGGCC CTCAGCGGAT TGGTAGGAAG TACAAAAAAG TCCGATTTAT2041 GGCATACACA GATGAAACCT TTAAGACTCG TGAAGCTATT CAGCATGAAT CAGGAATCTT2101 GGGACCTTTA CTTTATGGGG AAGTTGGAGA CACACTGTTG ATTATATTTA AGAATCAAGC2161 AAGCAGACCA TATAACATCT ACCCTCACGG AATCACTGAT GTCCGTCCTT TGTATTCAAG2221 GAGATTACCA AAAGGTGTAA AACATTTGAA GGATTTTCCA ATTCTGCCAG GAGAAATATT2281 CAAATATAAA TGGACAGTGA CTGTAGAAGA TGGGCCAACT AAATCAGATC CTCGGTGCCT2341 GACCCGCTAT TACTCTAGTT TCGTTAATAT GGAGAGAGAT CTAGCTTCAG GACTCATTGG2401 CCCTCTCCTC ATCTGCTACA AAGAATCTGT AGATCAAAGA GGAAACCAGA TAATGTCAGA2461 CAAGAGGAAT GTCATCCTGT TTTCTGTATT TGATGAGAAC CGAAGCTGGT ACCTCACAGA2521 GAATATACAA CGCTTTCTCC CCAATCCAGC TGGAGTGCAG CTTGAGGATC CAGAGTTCCA2581 AGCCTCCAAC ATCATGCACA GCATCAATGG CTATGTTTTT GATAGTTTGC AGTTGTCAGT2641 TTGTTTGCAT GAGGTGGCAT ACTGGTACAT TCTAAGCATT GGAGCACAGA CTGACTTCCT2701 TTCTGTCTTC TTCTCTGGAT ATACCTTCAA ACACAAAATG GTCTATGAAG ACACACTCAC2761 CCTATTCCCA TTCTCAGGAG AAACTGTCTT CATGTCGATG GAAAACCCAG GTCTATGGAT2821 TCTGGGGTGC CACAACTCAG ACTTTCGGAA CAGAGGCATG ACCGCCTTAC TGAAGGTTTC2881 TAGTTGTGAC AAGAACACTG GTGATTATTA CGAGGACAGT TATGAAGATA TTTCAGCATA2941 CTTGCTGAGT AAAAACAATG CCATTGAACC AAGAAGCTTC TCCCAGAATT CAAGACACCC3001 TAGCACTAGG CAAAAGCAAT TTAATGCCAC CACAATTCCA GAAAATGACA TAGAGAAGAC3061 TGACCCTTGG TTTGCACACA GAACACCTAT GCCTAAAATA CAAAATGTCT CCTCTAGTGA3121 TTTGTTGATG CTCTTGCGAC AGAGTCCTAC TCCACATGGG CTATCCTTAT CTGATCTCCA3241 AGAAGCCAAA TATGAGACTT TTTCTGATGA TCCATCACCT GGAGCAATAG ACAGTAATAA3301 CAGCCTGTCT GAAATGACAC ACTTCAGGCC ACAGCTCCAT CACAGTGGGG ACATGGTATT3361 TACCCCTGAG TCAGGCCTCC AATTAAGATT AAATGAGAAA CTGGGGACAA CTGCAGCAAC3421 AGAGTTGAAG AAACTTGATT TCAAAGTTTC TAGTACATCA AATAATCTGA TTTCAACAAT3481 TCCATCAGAC AATTTGGCAG CAGGTACTGA TAATACAAGT TCCTTAGGAC CCCCAAGTAT3541 GCCAGTTCAT TATGATAGTC AATTAGATAC CACTCTATTT GGCAAAAAGT CATCTCCCCT3601 TACTGAGTCT GGTGGACCTC TGAGCTTGAG TGAAGAAAAT AATGATTCAA AGTTGTTAGA3661 ATCAGGTTTA ATGAATAGCC AAGAAAGTTC ATGGGGAAAA AATGTATCGT CAACAGAGAG3721 TGGTAGGTTA TTTAAAGGGA AAAGAGCTCA TGGACCTGCT TTGTTGACTA AAGATAATGC3781 CTTATTCAAA GTTAGCATCT CTTTGTTAAA GACAAACAAA ACTTCCAATA ATTCAGCAAC3841 TAATAGAAAG ACTCACATTG ATGGCCCATC ATTATTAATT GAGAATAGTC CATCAGTCTG3901 GCAAAATATA TTAGAAAGTG ACACTGAGTT TAAAAAAGTG ACACCTTTGA TTCATGACAG3961 AATGCTTATG GACAAAAATG CTACAGCTTT GAGGCTAAAT CATATGTCAA ATAAAACTAC4021 TTCATCAAAA AACATGGAAA TGGTCCAACA GAAAAAAGAG GGCCCCATTC CACCAGATGC4081 ACAAAATCCA GATATGTCGT TCTTTAAGAT GCTATTCTTG CCAGAATCAG CAAGGTGGAT4141 ACAAAGGACT CATGGAAAGA ACTCTCTGAA CTCTGGGCAA GGCCCCAGTC CAAAGCAATT4201 AGTATCCTTA GGACCAGAAA AATCTGTGGA AGGTCAGAAT TTCTTGTCTG AGAAAAACAA4261 AGTGGTAGTA GGAAAGGGTG AATTTACAAA GGACGTAGGA CTCAAAGAGA TGGTTTTTCC4321 AAGCAGCAGA AACCTATTTC TTACTAACTT GGATAATTTA CATGAAAATA ATACACACAA4381 TCAAGAAAAA AAAATTCAGG AAGAAATAGA AAAGAAGGAA ACATTAATCC AAGAGAATGT4441 AGTTTTGCCT CAGATACATA CAGTGACTGG CACTAAGAAT TTCATGAAGA ACCTTTTCTT4501 ACTGAGCACT AGGCAAAATG TAGAAGGTTC ATATGACGGG GCATATGCTC CAGTACTTCA4561 AGATTTTAGG TCATTAAATG ATTCAACAAA TAGAACAAAG AAACACACAG CTCATTTCTC4621 AAAAAAAGGG GAGGAAGAAA ACTTGGAAGG CTTGGGAAAT CAAACCAAGC AAATTGTAGA4681 GAAATATGCA TGCACCACAA GGATATCTCC TAATACAAGC CAGCAGAATT TTGTCACGCA4741 ACGTAGTAAG AGAGCTTTGA AACAATTCAG ACTCCCACTA GAAGAAACAG AACTTGAAAA1801 AAGGATAATT GTGGATGACA CCTCAACCCA GTGGTCCAAA AACATGAAAC ATTTGACCCC4861 GAGCACCCTC ACACAGATAG ACTACAATGA GAAGGAGAAA GGGGCCATTA CTCAGTCTCC4921 CTTATCAGAT TGCCTTACGA GGAGTCATAG CATCCCTCAA GCAAATAGAT CTCCATTACC4981 CATTGCAAAG GTATCATCAT TTCCATCTAT TAGACCTATA TATCTGACCA GGGTCCTATT5041 CCAAGACAAC TCTTCTCATC TTCCAGCAGC ATCTTATAGA AAGAAAGATT CTGGGGTCCA5101 AGAAAGCAGT CATTTCTTAC AAGGAGCCAA AAAAAATAAC CTTTCTTTAG CCATTCTAAC5161 CTTGGAGATG ACTGGTGATC AAAGAGAGGT TGGCTCCCTG GGGACAAGTG CCACAAATTC5221 AGTCACATAC AAGAAAGTTG AGAACACTGT TCTCCCGAAA CCAGACTTGC CCAAAACATC5281 TGGCAAAGTT GAATTGCTTC CAAAAGTTCA CATTTATCAG AAGGACCTAT TCCCTACGGA5341 AACTAGCAAT GGGTCTCCTG GCCATCTGGA TCTCGTGGAA GGGAGCCTTC TTCAGGGAAC5401 AGAGGGAGCG ATTAAGTGGA ATGAAGCAAA CAGACCTGGA AAAGTTCCCT TTCTGAGAGT5461 AGCAACAGAA AGCTCTGCAA AGACTCCCTC CAAGCTATTG GATCCTCTTG CTTGGGATAA5521 CCACTATGGT ACTCAGATAC CAAAAGAAGA GTGGAAATCC CAAGAGAAGT CACCAGAAAA5581 AACAGCTTTT AAGAAAAAGG ATACCATTTT GTCCCTGAAC GCTTGTGAAA GCAATCATGC5641 AATAGCAGCA ATAAATGAGG GACAAAATAA GCCCGAAATA GAAGTCACCT GGGCAAAGCA5701 AGGTAGGACT GAAAGGCTGT GCTCTCAAAA CCCACCAGTC TTGAAACGCC ATCAACGGGA5761 AATAACTCGT ACTACTCTTC AGTCAGATCA AGAGGAAATT GACTATGATG ATACCATATC5851 AGTTGAAATG AAGAAGGAAG ATTTTGACAT TTATGATGAG GATGAAAATC AGAGCCCCCG5881 CAGCTTTCAA AAGAAAACAC GACACTATTT TATTGCTGCA GTGGAGAGGC TCTGGGATTA5941 TGGGATGAGT AGCTCCCCAC ATGTTCTAAG AAACAGGGCT CAGAGTGGCA GTGTCCCTCA6001 TGGAGAACTA AATGAACATT TGGGACTCCT GGGGCCATAT ATAAGAGCAG AAGTTGAAGA6061 TAATATCATG GTAACTTTCA GAAATCAGGC CTCTCGTCCC TATTCCTTCT ATTCTAGCCT6121 TATTTCTTAT GAGGAAGATC AGAGGCAAGG AGCAGAACCT AGAAAAAACT TTGTCAAGCC6181 TAATGAAACC AAAACTTACT TTTGGAAAGT GCAACATCAT ATGGCACCCA CTAAAGATGA6241 GTTTGACTGC AAAGCCTGGG CTTATTTCTC TGATGTTGAC CTGGAAAAAG ATGTGCACTC6301 AGGCCTGATT GGACCCCTTC TGGTCTGCCA CACTAACACA CTGAACCCTG CTCATGGGAG6361 ACAAGTGACA GTACAGGAAT TTGCTCTGTT TTTCACCATC TTTGATGAGA CCAAAAGCTG6421 GTACTTCACT GAAAATATGG AAAGAAACTG CAGGGCTCCC TGCAATATCC AGATGGAAGA6481 TCCCACTTTT AAAGAGAATT ATCGCTTCCA TGCAATCAAT GGCTACATAA TGGATACACT6541 ACCTGGCTTA GTAATGGCTC AGGATCAAAG GATTCGATGG TATCTGCTCA GCATGGGCAG6601 CAATGAAAAC ATCCATTCTA TTCATTTCAG TGGACATGTG TTCACTGTAC GAAAAAAAGA6661 GGAGTATAAA ATGGCACTGT ACAATCTCTA TCCAGGTGTT TTTGAGACAG TGGAAATGTT6721 ACCATCCAAA GCTGGAATTT GGCGGGTGGA ATGCCTTATT GGCGAGCATC TACATGCTGG6781 GATGAGCACA CTTTTTCTGG TGTACAGCAA TAAGTGTCAG ACTCCCCTGG GAATGGCTTC6841 TGGACACATT AGAGATTTTC AGATTACAGC TTCAGGACAA TATGGACAGT GGGCCCCAAA6901 GCTGGCCAGA CTTCATTATT CCGGATCAAT CAATGCCTGG AGCACCAAGG AGCCCTTTTC6961 TTGGATCAAG GTGGATCTGT TGGCACCAAT GATTATTCAC GGCATCAAGA CCCAGGGTGC7021 CCGTCAGAAG TTCTCCAGCC TCTACATCTC TCAGTTTATC ATCATGTATA GTCTTGATGG7081 GAAGAAGTGG CAGACTTATC GAGGAAATTC CACTGGAACC TTAATGGTCT TCTTTGGCAA7141 TGTGGATTCA TCTGGGATAA AACACAATAT TTTTAACCCT CCAATTATTG CTCGATACAT7201 CCGTTTGCAC CCAACTCATT ATAGCATTCG CAGCACTCTT CGCATGGAGT TGATGGGCTG7261 TGATTTAAAT AGTTGCAGCA TGCCATTGGG AATGGAGAGT AAAGCAATAT CAGATGCACA7321 GATTACTGCT TCATCCTACT TTACCAATAT GTTTGCCACC TGGTCTCCTT CAAAAGCTCG7381 ACTTCACCTC CAAGGGAGGA GTAATGCCTG GAGACCTCAG GTGAATAATC CAAAAGAGTG7441 GCTGCAAGTG GACTTCCAGA AGACAATGAA AGTCACAGGA GTAACTACTC AGGGAGTAAA7501 ATCTCTGCTT ACCAGCATGT ATGTGAAGGA GTTCCTCATC TCCAGCAGTC AAGATGGCCA7561 TCAGTGGACT CTCTTTTTTC AGAATGGCAA AGTAAAGGTT TTTCAGGGAA ATCAAGACTC7621 CTTCACACCT GTGGTGAACT CTCTAGACCC ACCGTTACTG ACTCGCTACC TTCGAATTCA7681 CCCCCAGAGT TGGGTGCACC AGATTGCCCT GAGGATGGAG GTTCTGGGCT GCGAGGCACA7741 GGACCTCTAC *The underlined nucleic acids encode a signal peptide.

FVIII polypeptides include full-length FVIII, full-length FVIII minusMet at the N-terminus, mature FVIII (minus the signal sequence), matureFVIII with an additional Met at the N-terminus, and/or FVIII with a fullor partial deletion of the B domain. In certain embodiments, FVIIIvariants include B domain deletions, whether partial or full deletions.

The human FVIII gene was isolated and expressed in mammalian cells(Toole, J. J., et al., Nature 312:342-347 (1984); Gitschier, J., et al.,Nature 312:326-330 (1984); Wood, W. I., et al., Nature 312:330-337(1984); Vehar, G. A., et al., Nature 312:337-342 (1984); WO 87/04187; WO88/08035; WO 88/03558; and U.S. Pat. No. 4,757,006). The FVIII aminoacid sequence was deduced from cDNA as shown in U.S. Pat. No. 4,965,199.In addition, partially or fully B-domain deleted FVIII is shown in U.S.Pat. Nos. 4,994,371 and 4,868,112. In some embodiments, the human FVIIIB-domain is replaced with the human Factor V B-domain as shown in U.S.Pat. No. 5,004,803. The cDNA sequence encoding human Factor VIII andamino acid sequence are shown in SEQ ID NOs: 17 and 16, respectively, ofUS Application Publ. No. 2005/0100990.

The porcine FVIII sequence is published in Toole, J. J., et al., Proc.Natl. Acad. Sci. USA 83:5939-5942 (1986). Further, the complete porcinecDNA sequence obtained from PCR amplification of FVIII sequences from apig spleen cDNA library has been reported in Healey, J. F., et al.,Blood 88:4209-4214 (1996). Hybrid human/porcine FVIII havingsubstitutions of all domains, all subunits, and specific amino acidsequences were disclosed in U.S. Pat. No. 5,364,771 by Lollar and Runge,and in WO 93/20093. More recently, the nucleotide and correspondingamino acid sequences of the A1 and A2 domains of porcine FVIII and achimeric FVIII with porcine A1 and/or A2 domains substituted for thecorresponding human domains were reported in WO 94/11503. U.S. Pat. No.5,859,204, Lollar, J. S., also discloses the porcine cDNA and deducedamino acid sequences. U.S. Pat. No. 6,458,563 discloses aB-domain-deleted porcine FVIII.

U.S. Pat. No. 5,859,204 to Lollar, J. S. reports functional mutants ofFVIII having reduced antigenicity and reduced immunoreactivity. U.S.Pat. No. 6,376,463 to Lollar, J. S. also reports mutants of FVIII havingreduced immunoreactivity. US Appl. Publ. No. 2005/0100990 to Saenko etal. reports functional mutations in the A2 domain of FVIII.

In one embodiment, the FVIII (or FVIII portion of a chimeric protein)may be at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or100% identical to a FVIII amino acid sequence of amino acids 1 to 1438of SEQ ID NO: 18 or amino acids 1 to 2332 of SEQ ID NO: 16 (without asignal sequence) or a FVIII amino acid sequence of amino acids −19 to1438 of SEQ ID NO: 15 and SEQ ID NO: 18 or amino acids −19 to 2332 ofSEQ ID NO: 15 and SEQ ID NO: 16 (with a signal sequence), wherein theFVIII has a clotting activity, e.g., activates Factor IX as a cofactorto convert Factor X to activated Factor X. The FVIII (or FVIII portionof a chimeric protein) may be identical to a FVIII amino acid sequenceof amino acids 1 to 1438 of SEQ ID NO: 18 or amino acids 1 to 2332 ofSEQ ID NO: 16 (without a signal sequence). The FVIII may furthercomprise a signal sequence.

The “B-domain” of FVIII, as used herein, is the same as the B-domainknown in the art that is defined by internal amino acid sequenceidentity and sites of proteolytic cleavage, e.g., residuesSer741-Arg1648 of full-length human FVIII. The other human FVIII domainsare defined by the following amino acid residues: A1, residuesAla1-Arg372; A2, residues Ser373-Arg740; A3, residues Ser1690-Asn2019;C1, residues Lys2020-Asn2172; C2, residues Ser2173-Tyr2332. The A3-C1-C2sequence includes residues Ser1690-Tyr2332. The remaining sequence,residues Glu1649-Arg1689, is usually referred to as the a3 acidicregion. The locations of the boundaries for all of the domains,including the B-domains, for porcine, mouse and canine FVIII are alsoknown in the art. In one embodiment, the B domain of FVIII is deleted(“B-domain-deleted factor VIII” or “BDD FVIII”). An example of a BDDFVIII is REFACTO® (recombinant BDD FVIII), which has the same sequenceas the Factor VIII portion of the sequence in Table 4. (BDD FVIII heavychain is double underlined; B domain is italicized; and BDD FVIII lightchain is in plain text).

TABLE 4 BDD FVIII (SEQ ID NO: 18)

SPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKWFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPWNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY

TABLE 5Nucleotide Sequence Encoding BDD FVIII (SEQ ID NO: 19)* TGCTTCTTTC 661                                          A TGCAAATAGA GCTCTCCACC 721TGTGCCTTTT GCGATTCTGC TTTAGTGCCA CCAGAAGATA CTACCTGGGT GCAGTGGAAC 781TGTCATGGGA CTATATGCAA AGTGATCTCG GTGAGCTGCC TGTGGACGCA AGATTTCCTC 841CTAGAGTGCC AAAATCTTTT CCATTCAACA CCTCAGTCGT GTACAAAAAG ACTCTGTTTG 901TAGAATTCAC GGATCACCTT TTCAACATCG CTAAGCCAAG GCCACCCTGG ATGGGTCTGC 961TAGGTCCTAC CATCCAGGCT GAGGTTTATG ATACAGTGGT CATTACACTT AAGAACATGG 101CTTCCCATCC TGTCAGTCTT CATGCTGTTG GTGTATCCTA CTGGAAAGCT TCTGAGGGAG 1081CTGAATATGA TGATCAGACC AGTCAAAGGG AGAAAGAAGA TGATAAAGTC TTCCCTGGTG 1141GAAGCCATAC ATATGTCTGG CAGGTCCTGA AAGAGAATGG TCCAATGGCC TCTGACCCAC 1201TGTGCCTTAC CTACTCATAT CTTTCTCATG TGGACCTGGT AAAAGACTTG AATTCAGGCC 1261TCATTGGAGC CCTACTAGTA TGTAGAGAAG GGAGTCTGGC CAAGGAAAAG ACACAGACCT 1321TGCACAAATT TATACTACTT TTTGCTGTAT TTGATGAAGG GAAAAGTTGG CACTCAGAAA 1381CAAAGAACTC CTTGATGCAG GATAGGGATG CTGCATCTGC TCGGGCCTGG CCTAAAATGC 1441ACACAGTCAA TGGTTATGTA AACAGGTCTC TGCCAGGTCT GATTGGATGC CACAGGAAAT 1501CAGTCTATTG GCATGTGATT GGAATGGGCA CCACTCCTGA AGTGCACTCA ATATTCCTCG 1561AAGGTCACAC ATTTCTTGTG AGGAACCATC GCCAGGCGTC CTTGGAAATC TCGCCAATAA 1621CTTTCCTTAC TGCTCAAACA CTCTTGATGG ACCTTGGACA GTTTCTACTG TTTTGTCATA 1681TCTCTTCCCA CCAACATGAT GGCATGGAAG CTTATGTCAA AGTAGACAGC TGTCCAGAGG 1741AACCCCAACT ACGAATGAAA AATAATGAAG AAGCGGAAGA CTATGATGAT GATCTTACTG 1801ATTCTGAAAT GGATGTGGTC AGGTTTGATG ATGACAACTC TCCTTCCTTT ATCCAAATTC 1861GCTCAGTTGC CAAGAAGCAT CCTAAAACTT GGGTACATTA CATTGCTGCT GAAGAGGAGG 1921ACTGGGACTA TGCTCCCTTA GTCCTCGCCC CCGATGACAG AAGTTATAAA AGTCAATATT 1981TGAACAATGG CCCTCAGCGG ATTGGTAGGA AGTACAAAAA AGTCCGATTT ATGGCATACA 2041CAGATGAAAC CTTTAAGACT CGTGAAGCTA TTCAGCATGA ATCAGGAATC TTGGGACCTT 2101TACTTTATGG GGAAGTTGGA GACACACTGT TGATTATATT TAAGAATCAA GCAAGCAGAC 2161CATATAACAT CTACCCTCAC GGAATCACTG ATGTCCGTCC TTTGTATTCA AGGAGATTAC 2221CAAAAGGTGT AAAACATTTG AAGGATTTTC CAATTCTGCC AGGAGAAATA TTCAAATATA 2281AATGGACAGT GACTGTAGAA GATGGGCCAA CTAAATCAGA TCCTCGGTGC CTGACCCGCT 2341ATTACTCTAG TTTCGTTAAT ATGGAGAGAG ATCTAGCTTC AGGACTCATT GGCCCTCTCC 2401TCATCTGCTA CAAAGAATCT GTAGATCAAA GAGGAAACCA GATAATGTCA GACAAGAGGA 2461ATGTCATCCT GTTTTCTGTA TTTGATGAGA ACCGAAGCTG GTACCTCACA GAGAATATAC 2521AACGCTTTCT CCCCAATCCA GCTGGAGTGC AGCTTGAGGA TCCAGAGTTC CAAGCCTCCA 2581ACATCATGCA CAGCATCAAT GGCTATGTTT TTGATAGTTT GCAGTTGTCA GTTTGTTTGC 2641ATGAGGTGGC ATACTGGTAC ATTCTAAGCA TTGGAGCACA GACTGACTTC CTTTCTGTCT 2701TCTTCTCTGG ATATACCTTC AAACACAAAA TGGTCTATGA AGACACACTC ACCCTATTCC 2761CATTCTCAGG AGAAACTGTC TTCATGTCGA TGGAAAACCC AGGTCTATGG ATTCTGGGGT 2821GCCACAACTC AGACTTTCGG AACAGAGGCA TGACCGCCTT ACTGAAGGTT TCTAGTTGTG 2881ACAAGAACAC TGGTGATTAT TACGAGGACA GTTATGAAGA TATTTCAGCA TACTTGCTGA 2941GTAAAAACAA TGCCATTGAA CCAAGAAGCT TCTCTCAAAA CCCACCAGTC TTGAAACGCC 3001ATCAACGGGA AATAACTCGT ACTACTCTTC AGTCAGATCA AGAGGAAATT GACTATGATG 3061ATACCATATC AGTTGAAATG AAGAAGGAAG ATTTTGACAT TTATGATGAG GATGAAAATC 3121AGAGCCCCCG CAGCTTTCAA AAGAAAACAC GACACTATTT TATTGCTGCA GTGGAGAGGC 3181TCTGGGATTA TGGGATGAGT AGCTCCCCAC ATGTTCTAAG AAACAGGGCT CAGAGTGGCA 3241GTGTCCCTCA GTTCAAGAAA GTTGTTTTCC AGGAATTTAC TGATGGCTCC TTTACTCAGC 3301CCTTATACCG TGGAGAACTA AATGAACATT TGGGACTCCT GGGGCCATAT ATAAGAGCAG 3361AAGTTGAAGA TAATATCATG GTAACTTTCA GAAATCAGGC CTCTCGTCCC TATTCCTTCT 3421ATTCTAGCCT TATTTCTTAT GAGGAAGATC AGAGGCAAGG AGCAGAACCT AGAAAAAACT 3481TTGTCAAGCC TAATGAAACC AAAACTTACT TTTGGAAAGT GCAACATCAT ATGGCACCCA 3541CTAAAGATGA GTTTGACTGC AAAGCCTGGG CTTATTTCTC TGATGTTGAC CTGGAAAAAG 3601ATGTGCACTC AGGCCTGATT GGACCCCTTC TGGTCTGCCA CACTAACACA CTGAACCCTG 3661CTCATGGGAG ACAAGTGACA GTACAGGAAT TTGCTCTGTT TTTCACCATC TTTGATGAGA 3721CCAAAAGCTG GTACTTCACT GAAAATATGG AAAGAAACTG CAGGGCTCCC TGCAATATCC 3781AGATGGAAGA TCCCACTTTT AAAGAGAATT ATCGCTTCCA TGCAATCAAT GGCTACATAA 3841TGGATACACT ACCTGGCTTA GTAATGGCTC AGGATCAAAG GATTCGATGG TATCTGCTCA 3901GCATGGGCAG CAATGAAAAC ATCCATTCTA TTCATTTCAG TGGACATGTG TTCACTGTAC 3961GAAAAAAAGA GGAGTATAAA ATGGCACTGT ACAATCTCTA TCCAGGTGTT TTTGAGACAG 4021TGGAAATGTT ACCATCCAAA GCTGGAATTT GGCGGGTGGA ATGCCTTATT GGCGAGCATC 4081TACATGCTGG GATGAGCACA CTTTTTCTGG TGTACAGCAA TAAGTGTCAG ACTCCCCTGG 4141GAATGGCTTC TGGACACATT AGAGATTTTC AGATTACAGC TTCAGGACAA TATGGACAGT 4201GGGCCCCAAA GCTGGCCAGA CTTCATTATT CCGGATCAAT CAATGCCTGG AGCACCAAGG 4261AGCCCTTTTC TTGGATCAAG GTGGATCTGT TGGCACCAAT GATTATTCAC GGCATCAAGA 4321CCCAGGGTGC CCGTCAGAAG TTCTCCAGCC TCTACATCTC TCAGTTTATC ATCATGTATA 4381GTCTTGATGG GAAGAAGTGG CAGACTTATC GAGGAAATTC CACTGGAACC TTAATGGTCT 4441TCTTTGGCAA TGTGGATTCA TCTGGGATAA AACACAATAT TTTTAACCCT CCAATTATTG 4501CTCGATACAT CCGTTTGCAC CCAACTCATT ATAGCATTCG CAGCACTCTT CGCATGGAGT 4561TGATGGGCTG TGATTTAAAT AGTTGCAGCA TGCCATTGGG AATGGAGAGT AAAGCAATAT 4621CAGATGCACA GATTACTGCT TCATCCTACT TTACCAATAT GTTTGCCACC TGGTCTCCTT 4681CAAAAGCTCG ACTTCACCTC CAAGGGAGGA GTAATGCCTG GAGACCTCAG GTGAATAATC 4741CAAAAGAGTG GCTGCAAGTG GACTTCCAGA AGACAATGAA AGTCACAGGA GTAACTACTC 4801AGGGAGTAAA ATCTCTGCTT ACCAGCATGT ATGTGAAGGA GTTCCTCATC TCCAGCAGTC 4861AAGATGGCCA TCAGTGGACT CTCTTTTTTC AGAATGGCAA AGTAAAGGTT TTTCAGGGAA 4921ATCAAGACTC CTTCACACCT GTGGTGAACT CTCTAGACCC ACCGTTACTG ACTCGCTACC 4981TTCGAATTCA CCCCCAGAGT TGGGTGCACC AGATTGCCCT GAGGATGGAG GTTCTGGGCT 5041GCGAGGCACA GGACCTCTAC *The underlined nucleic acids encode a signalpeptide.

A “B-domain-deleted FVIII” may have the full or partial deletionsdisclosed in U.S. Pat. Nos. 6,316,226, 6,346,513, 7,041,635, 5,789,203,6,060,447, 5,595,886, 6,228,620, 5,972,885, 6,048,720, 5,543,502,5,610,278, 5,171,844, 5,112,950, 4,868,112, and 6,458,563. In someembodiments, a B-domain-deleted FVIII sequence of the present inventioncomprises any one of the deletions disclosed at col. 4, line 4 to col.5, line 28 and Examples 1-5 of U.S. Pat. No. 6,316,226 (also in U.S.Pat. No. 6,346,513). In another embodiment, a B-domain deleted FactorVIII is the 5743/Q1638 B-domain deleted Factor VIII (SQ BDD FVIII)(e.g., Factor VIII having a deletion from amino acid 744 to amino acid1637, e.g., Factor VIII having amino acids 1-743 and amino acids1638-2332 of SEQ ID NO: 16, i.e., SEQ ID NO: 18). In some embodiments, aB-domain-deleted FVIII of the present invention has a deletion disclosedat col. 2, lines 26-51 and examples 5-8 of U.S. Pat. No. 5,789,203 (alsoU.S. Pat. Nos. 6,060,447, 5,595,886, and 6,228,620). In someembodiments, a B-domain-deleted Factor VIII has a deletion described incol. 1, lines 25 to col. 2, line 40 of U.S. Pat. No. 5,972,885; col. 6,lines 1-22 and example 1 of U.S. Pat. No. 6,048,720; col. 2, lines 17-46of U.S. Pat. No. 5,543,502; col. 4, line 22 to col. 5, line 36 of U.S.Pat. No. 5,171,844; col. 2, lines 55-68, FIG. 2 , and example 1 of U.S.Pat. No. 5,112,950; col. 2, line 2 to col. 19, line 21 and table 2 ofU.S. Pat. No. 4,868,112; col. 2, line 1 to col. 3, line 19, col. 3, line40 to col. 4, line 67, col. 7, line 43 to col. 8, line 26, and col. 11,line 5 to col. 13, line 39 of U.S. Pat. No. 7,041,635; or col. 4, lines25-53, of U.S. Pat. No. 6,458,563.

In some embodiments, a B-domain-deleted FVIII has a deletion of most ofthe B domain, but still contains amino-terminal sequences of the Bdomain that are essential for in vivo proteolytic processing of theprimary translation product into two polypeptide chain, as disclosed inWO 91/09122. In some embodiments, a B-domain-deleted FVIII isconstructed with a deletion of amino acids 747-1638, i.e., virtually acomplete deletion of the B domain. Hoeben R. C., et al. J. Biol. Chem.265 (13): 7318-7323 (1990). A B-domain-deleted Factor VIII may alsocontain a deletion of amino acids 771-1666 or amino acids 868-1562 ofFVIII. Meulien P., et al. Protein Eng. 2(4): 301-6 (1988). Additional Bdomain deletions that are part of the invention include: deletion ofamino acids 982 through 1562 or 760 through 1639 (Toole et al., Proc.Natl. Acad. Sci. U.S.A. (1986) 83, 5939-5942)), 797 through 1562 (Eaton,et al. Biochemistry (1986) 25:8343-8347)), 741 through 1646 (Kaufman(PCT published application No. WO 87/04187)), 747-1560 (Sarver, et al.,DNA (1987) 6:553-564)), 741 through 1648 (Pasek (PCT application No.88/00831)), or 816 through 1598 or 741 through 1648 (Lagner (BehringInst. Mitt. (1988) No 82:16-25, EP 295597)). In other embodiments, BDDFVIII includes a FVIII polypeptide containing fragments of the B-domainthat retain one or more N-linked glycosylation sites, e.g., residues757, 784, 828, 900, 963, or optionally 943, which correspond to theamino acid sequence of the full-length FVIII sequence. Examples of theB-domain fragments include 226 amino acids or 163 amino acids of theB-domain as disclosed in Miao, H. Z., et al., Blood 103(a): 3412-3419(2004), Kasuda, A, et al., J. Thromb. Haemost. 6: 1352-1359 (2008), andPipe, S. W., et al., J Thromb. Haemost. 9: 2235-2242 (2011) (i.e., thefirst 226 amino acids or 163 amino acids of the B domain are retained).In some embodiments, the FVIII with a partial B-domain is FVIII198 (SEQID NO: 105). FVIII198 is a partial B-domain containing single chainFVIIIFc molecule-226N6. 226 represents the N-terminus 226 amino acid ofthe FVIII B-domain, and N6 represents six N-glycosylation sites in theB-domain. In still other embodiments, BDD FVIII further comprises apoint mutation at residue 309 (from Phe to Ser) to improve expression ofthe BDD FVIII protein. See Miao, H. Z., et al., Blood 103(a): 3412-3419(2004). In still other embodiments, the BDD FVIII includes a FVIIIpolypeptide containing a portion of the B-domain, but not containing oneor more furin cleavage sites (e.g., Arg1313 and Arg 1648). See Pipe, S.W., et al., J Thromb. Haemost. 9: 2235-2242 (2011). Each of theforegoing deletions may be made in any FVIII sequence.

A FVIII protein useful in the present invention can include FVIII havingone or more additional heterologous sequences or chemical or physicalmodifications therein, which do not affect the FVIII coagulationactivity. Such heterologous sequences or chemical or physicalmodifications can be fused to the C-terminus or N-terminus of the FVIIIprotein or inserted between one or more of the two amino acid residuesin the FVIII protein. Such insertions in the FVIII protein do not affectthe FVIII coagulation activity or FVIII function. In one embodiment, theinsertions improve pharmacokinetic properties of the FVIII protein(e.g., half-life). In another embodiment, the insertions can be morethan two, three, four, five, or six sites.

In one embodiment, FVIII is cleaved right after Arginine at amino acid1648 (in full-length Factor VIII or SEQ ID NO: 16), amino acid 754 (inthe 5743/Q1638 B-domain deleted Factor VIII or SEQ ID NO: 16), or thecorresponding Arginine residue (in other variants), thereby resulting ina heavy chain and a light chain. In another embodiment, FVIII comprisesa heavy chain and a light chain, which are linked or associated by ametal ion-mediated non-covalent bond.

In other embodiments, FVIII is a single chain FVIII that has not beencleaved right after Arginine at amino acid 1648 (in full-length FVIII orSEQ ID NO: 16), amino acid 754 (in the 5743/Q1638 B-domain-deleted FVIIIor SEQ ID NO: 18), or the corresponding Arginine residue (in othervariants). A single chain FVIII may comprise one or more amino acidsubstitutions. In one embodiment, the amino acid substitution is at aresidue corresponding to residue 1648, residue 1645, or both offull-length mature Factor VIII polypeptide (SEQ ID NO: 16) or residue754, residue 751, or both of SQ BDD Factor VIII (SEQ ID NO: 18). Theamino acid substitution can be any amino acids other than Arginine,e.g., isoleucine, leucine, lysine, methionine, phenylalanine, threonine,tryptophan, valine, alanine, asparagine, aspartic acid, cysteine,glutamic acid, glutamine, glycine, proline, selenocysteine, serine,tyrosine, histidine, ornithine, pyrrolysine, or taurine.

FVIII can further be cleaved by thrombin and then activated as FVIIIa,serving as a cofactor for activated Factor IX (FIXa). And the activatedFIX together with activated FVIII forms a Xase complex and convertsFactor X to activated Factor X (FXa). For activation, FVIII is cleavedby thrombin after three Arginine residues, at amino acids 372, 740, and1689 (corresponding to amino acids 372, 740, and 795 in the B-domaindeleted FVIII sequence), the cleavage generating FVIIIa having the 50kDa A1, 43 kDa A2, and 73 kDa A3-C1-C2 chains. In one embodiment, theFVIII protein useful for the present invention is non-active FVIII. Inanother embodiment, the FVIII protein is an activated FVIII.

The protein having FVIII polypeptide linked to or associated with theVWF fragment can comprise a sequence at least 50%, 60%, 70%, 80%, 90%,95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 16 or 18,wherein the sequence has the FVIII clotting activity, e.g., activatingFactor IX as a cofactor to convert Factor X to activated Factor X (FXa).

“Hybrid” polypeptides and proteins, as used herein, means a combinationof a first polypeptide chain, e.g., the VWF fragment, optionally fusedto a first heterologous moiety, with a second polypeptide chain, e.g., aFVIII protein, optionally fused to a second heterologous moiety, therebyforming a heterodimer. In one embodiment, the first polypeptide and thesecond polypeptide in a hybrid are associated with each other viaprotein-protein interactions, such as charge-charge or hydrophobicinteractions. In another embodiment, the first polypeptide and thesecond polypeptide in a hybrid are associated with each other viadisulfide or other covalent bond(s). Hybrids are described, for example,in US 2004/101740 and US 2006/074199. The second polypeptide may be anidentical copy of the first polypeptide or a non-identical polypeptide.In one embodiment, the first polypeptide is a VWF fragment-Fc fusionprotein, and the second polypeptide is a polypeptide comprising,consisting essentially of, or consisting of an FcRn binding domain,wherein the first polypeptide and the second polypeptide are associatedwith each other. In another embodiment, the first polypeptide comprisesa VWF fragment-Fc fusion protein, and the second polypeptide comprisesFVIII-Fc fusion protein, making the hybrid a heterodimer. The firstpolypeptide and the second polypeptide can be associated through acovalent bond, e.g., a disulfide bond, between the first Fc region andthe second Fc region. The first polypeptide and the second polypeptidecan further be associated with each other by binding between the VWFfragment and the FVIII protein.

D) Linkers

The chimeric protein of the present invention further comprises alinker. One or more linkers can be present between any two proteins,e.g., between the adjunct moiety and the FVIII protein (sometimes alsoreferred to as “FVIII/AM linker”), between the VWF fragment and a firstheterologous moiety (sometime also referred to as “VWF linker”), e.g., afirst Fc region, between a FVIII protein and a second heterologousmoiety (sometimes also referred to as “FVIII linker”), e.g., a second Fcregion, between the VWF fragment and a FVIII protein (e.g., FVIII/AMlinker), between the VWF fragment and a second heterologous moiety,and/or between a FVIII protein and a first heterologous moiety. Each ofthe linkers can have the same or different sequence. In one embodiment,the linker is a polypeptide linker. In another embodiment, the linker isa non-polypeptide linker.

The linker useful in the present invention can comprise any organicmolecule. In one embodiment, the linker is a polymer, e.g., polyethyleneglycol (PEG) or hydroxyethyl starch (HES). In another embodiment, thelinker is an amino acid sequence (e.g., a polypeptide linker). Thepolypeptide linker can comprise at least about 10, 20, 30, 40, 50, 60,70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000,1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 aminoacids. The linker can comprise 1-5 amino acids, 1-10 amino acids, 1-20amino acids, 10-50 amino acids, 50-100 amino acids, 100-200 amino acids,200-300 amino acids, 300-400 amino acids, 400-500 amino acids, 500-600amino acids, 600-700 amino acids, 700-800 amino acids, 800-900 aminoacids, or 900-1000 amino acids.

Examples of polypeptide linkers are well known in the art. In oneembodiment, the linker comprises the sequence Gn. The linker cancomprise the sequence (GA)_(n). The linker can comprise the sequence(GGS)_(n). In other embodiments, the linker comprises (GGGS)_(n) (SEQ IDNO: 20). In still other embodiments, the linker comprises the sequence(GGS)_(n)(GGGGS)_(n) (SEQ ID NO: 21). In these instances, n may be aninteger from 1-100. In other instances, n may be an integer from 1-20,i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,or 20. Examples of linkers include, but are not limited to, GGG, SGGSGGS(SEQ ID NO: 22), GGSGGSGGSGGSGGG (SEQ ID NO: 23), GGSGGSGGGGSGGGGS (SEQID NO: 24), GGSGGSGGSGGSGGSGGS (SEQ ID NO: 25), GGGGSGGGGSGGGGS (SEQ IDNO: 26), the linkers in Table 13 (SEQ ID NOs: 92, 93, and 94), and thelinkers in Table 14A (SEQ ID NOs: 95, 96 and 97). The linker does noteliminate or diminish the VWF fragment activity or the clotting activityof Factor VIII. Optionally, the linker enhances the VWF fragmentactivity or the clotting activity of Factor VIII protein, e.g., byfurther diminishing the effects of steric hindrance and making the VWFfragment or Factor VIII portion more accessible to its target bindingsite.

In one embodiment, the linker useful for the chimeric protein is 15-25amino acids long. In another embodiment, the linker useful for thechimeric protein is 15-20 amino acids long. In some embodiments, thelinker for the chimeric protein is 10-25 amino acids long. In otherembodiments, the linker for the chimeric protein is 15 amino acids long.In still other embodiments, the linker for the chimeric protein is(GGGGS)_(n) (SEQ ID NO: 27) where G represents glycine, S representsserine and n is an integer from 1-20.

E) Cleavage Sites

The linker may also incorporate a moiety capable of being cleaved eitherchemically (e.g., hydrolysis of an ester bond), enzymatically (i.e.,incorporation of a protease cleavage sequence), or photolytically (e.g.,a chromophore such as 3-amino-3-(2-nitrophenyl) proprionic acid (ANP))in order to release one molecule from another.

In one embodiment, the linker is a cleavable linker. The cleavablelinkers can comprise one or more cleavage sites at the N-terminus orC-terminus or both. In another embodiment, the cleavable linker consistsessentially of or consists of one or more cleavable sites. In otherembodiments, the cleavable linker comprises heterologous amino acidlinker sequences described herein or polymers and one or more cleavablesites.

In certain embodiments, a cleavable linker comprises one or morecleavage sites that can be cleaved in a host cell (i.e., intracellularprocessing sites). Non limiting examples of the cleavage site includeRRRR (SEQ ID NO: 52), RKRRKR (SEQ ID NO: 53), and RRRRS (SEQ ID NO: 54).

In other embodiments, a cleavable linker comprises one or more cleavagesites that are cleaved by a protease after a chimeric protein comprisingthe cleavable linker is administered to a subject. In one embodiment,the cleavage site is cleaved by a protease selected from the groupconsisting of factor XIa, factor XIIa, kallikrein, factor VIIa, factorIXa, factor Xa, factor IIa (thrombin), Elastase-2, MMP-12, MMP-13,MMP-17, and MMP-20. In another embodiment, the cleavage site is selectedfrom the group consisting of a FXIa cleavage site (e.g., KLTR↓AET (SEQID NO: 29)), a FXIa cleavage site (e.g, DFTR↓VVG (SEQ ID NO: 30)), aFXIIa cleavage site (e.g., TMTR↓IVGG (SEQ ID NO: 31)), a Kallikreincleavage site (e.g., SPFR↓STGG (SEQ ID NO: 32)), a FVIIa cleavage site(e.g., LQVR↓IVGG (SEQ ID NO: 33)), a FIXa cleavage site (e.g., PLGR↓IVGG(SEQ ID NO: 34)), a FXa cleavage site (e.g., IEGR↓TVGG (SEQ ID NO: 35)),a FIIa (thrombin) cleavage site (e.g, LTPR↓SLLV (SEQ ID NO: 36)), aElastase-2 cleavage site (e.g, LGPV↓SGVP (SEQ ID NO: 37)), a Granzyme-Bcleavage (e.g, VAGD↓SLEE (SEQ ID NO: 38)), a MMP-12 cleavage site (e.g.,GPAG↓LGGA (SEQ ID NO: 39)), a MMP-13 cleavage site (e.g., GPAG↓LRGA (SEQID NO: 40)), a MMP-17 cleavage site (e.g., APLG↓LRLR (SEQ ID NO: 41)), aMMP-20 cleavage site (e.g., PALP↓LVAQ (SEQ ID NO: 42)), a TEV cleavagesite (e.g., ENLYFQ↓G (SEQ ID NO: 43)), a Enterokinase cleavage site(e.g., DDDK↓IVGG (SEQ ID NO: 44)), a Protease 3C (PRESCISSION™) cleavagesite (e.g., LEVLFQ↓GP (SEQ ID NO: 45)), and a Sortase A cleavage site(e.g., LPKT↓GSES) (SEQ ID NO: 46). In certain embodiments, the FXIacleavage sites include, but are not limited to, e.g., TQSFNDFTR (SEQ IDNO: 47) and SVSQTSKLTR (SEQ ID NO: 48). Non-limiting exemplary thrombincleavage sites include, e.g., DFLAEGGGVR (SEQ ID NO: 49), TTKIKPR (SEQID NO: 50), or LVPRG (SEQ ID NO: 55), and a sequence comprising,consisting essentially of, or consisting of ALRPR (e.g., ALRPRVVGGA (SEQID NO: 51)).

In a specific embodiment, the cleavage site is TLDPRSFLLRNPNDKYEPFWEDEEK(SEQ ID NO: 56).

Polynucleotides, Vectors, Host Cells, and Methods of Making

Also provided in the invention is a polynucleotide encoding a VWFfragment described herein, a chimeric protein comprising the VWFfragment and a heterologous moiety, a chimeric protein comprising aFVIII protein and an adjunct moiety, or a chimeric protein comprising aVWF fragment and a FVIII protein. When a VWF fragment is linked to aheterologous moiety or a FVIII protein in a chimeric protein as a singlepolypeptide chain, the invention is drawn to a polynucleotide encodingthe VWF fragment linked to the heterologous moiety or the FVIII protein.When the chimeric protein comprises a first and a second polypeptidechains, the first polypeptide chain comprising a VWF fragment and afirst heterologous moiety (e.g., a first Fc region) and the secondpolypeptide chain comprising a second heterologous moiety (e.g., asecond Fc region), wherein the first polypeptide chain and the secondpolypeptide chain are associated with each other, a polynucleotide cancomprise the first nucleotide sequence and the second nucleotidesequence. In one embodiment, the first nucleotide sequence and thesecond nucleotide sequence are on the same polynucleotide. In anotherembodiment, the first nucleotide sequence and the second nucleotidesequence are on two different polynucleotides (e.g., different vectors).In certain embodiments, the present invention is directed to a set ofpolynucleotides comprising a first nucleotide chain and a secondnucleotide chain, wherein the first nucleotide chain encodes the VWFfragment of the chimeric protein and the second nucleotide chain encodesthe FVIII protein.

In other embodiments, the set of the polynucleotides further comprisesan additional nucleotide chain (e.g., a second nucleotide chain when thechimeric polypeptide is encoded by a single polynucleotide chain or athird nucleotide chain when the chimeric protein is encoded by twopolynucleotide chains) which encodes a protein convertase. The proteinconvertase can be selected from the group consisting of proproteinconvertase subtilisin/kexin type 5 (PCSK5 or PC5), proprotein convertasesubtilisin/kexin type 7 (PCSK7 or PC5), a yeast Kex 2, proproteinconvertase subtilisin/kexin type 3 (PACE or PCSK3), and two or morecombinations thereof. In some embodiments, the protein convertase isPACE, PC5, or PC7. In a specific embodiment, the protein convertase isPC5 or PC7. See International Application no. PCT/US2011/043568, whichis incorporated herein by reference. In another embodiment, the proteinconvertase is PACE/Furin.

In certain embodiments, the invention includes a set of thepolynucleotides comprising a first nucleotide sequence encoding a VWFfragment comprising a D′ domain and a D3 domain of VWF, a secondnucleotide sequence encoding a FVIII protein, and a third nucleotidesequence encoding a D1 domain and D2 domain of VWF. In this embodiment,the D1 domain and D2 domain are separately expressed (not linked to theD′D3 domain of the VWF fragment) in order for the proper disulfide bondformation and folding of the D′D3 domains. The D1D2 domain expressioncan either be in cis or trans.

As used herein, an expression vector refers to any nucleic acidconstruct which contains the necessary elements for the transcriptionand translation of an inserted coding sequence, or in the case of an RNAviral vector, the necessary elements for replication and translation,when introduced into an appropriate host cell. Expression vectors caninclude plasmids, phagemids, viruses, and derivatives thereof.

Expression vectors of the invention will include polynucleotidesencoding the VWF fragment or the chimeric protein comprising the VWFfragment.

In one embodiment, a coding sequence for the VWF fragment, the secondheterologous moiety (e.g., a second Fc region), or the FVIII protein isoperably linked to an expression control sequence. As used herein, twonucleic acid sequences are operably linked when they are covalentlylinked in such a way as to permit each component nucleic acid sequenceto retain its functionality. A coding sequence and a gene expressioncontrol sequence are said to be operably linked when they are covalentlylinked in such a way as to place the expression or transcription and/ortranslation of the coding sequence under the influence or control of thegene expression control sequence. Two DNA sequences are said to beoperably linked if induction of a promoter in the 5′ gene expressionsequence results in the transcription of the coding sequence and if thenature of the linkage between the two DNA sequences does not (1) resultin the introduction of a frame-shift mutation, (2) interfere with theability of the promoter region to direct the transcription of the codingsequence, or (3) interfere with the ability of the corresponding RNAtranscript to be translated into a protein. Thus, a gene expressionsequence would be operably linked to a coding nucleic acid sequence ifthe gene expression sequence were capable of effecting transcription ofthat coding nucleic acid sequence such that the resulting transcript istranslated into the desired protein or polypeptide.

A gene expression control sequence as used herein is any regulatorynucleotide sequence, such as a promoter sequence or promoter-enhancercombination, which facilitates the efficient transcription andtranslation of the coding nucleic acid to which it is operably linked.The gene expression control sequence may, for example, be a mammalian orviral promoter, such as a constitutive or inducible promoter.Constitutive mammalian promoters include, but are not limited to, thepromoters for the following genes: hypoxanthine phosphoribosyltransferase (HPRT), adenosine deaminase, pyruvate kinase, beta-actinpromoter, and other constitutive promoters. Exemplary viral promoterswhich function constitutively in eukaryotic cells include, for example,promoters from the cytomegalovirus (CMV), simian virus (e.g., SV40),papilloma virus, adenovirus, human immunodeficiency virus (HIV), Roussarcoma virus, cytomegalovirus, the long terminal repeats (LTR) ofMoloney leukemia virus, and other retroviruses, and the thymidine kinasepromoter of herpes simplex virus. Other constitutive promoters are knownto those of ordinary skill in the art. The promoters useful as geneexpression sequences of the invention also include inducible promoters.Inducible promoters are expressed in the presence of an inducing agent.For example, the metallothionein promoter is induced to promotetranscription and translation in the presence of certain metal ions.Other inducible promoters are known to those of ordinary skill in theart.

In general, the gene expression control sequence shall include, asnecessary, 5′ non-transcribing and 5′ non-translating sequences involvedwith the initiation of transcription and translation, respectively, suchas a TATA box, capping sequence, CAAT sequence, and the like.Especially, such 5′ non-transcribing sequences will include a promoterregion which includes a promoter sequence for transcriptional control ofthe operably joined coding nucleic acid. The gene expression sequencesoptionally include enhancer sequences or upstream activator sequences asdesired.

Viral vectors include, but are not limited to, nucleic acid sequencesfrom the following viruses: retrovirus, such as Moloney murine leukemiavirus, Harvey murine sarcoma virus, murine mammary tumor virus, and Roussarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses;polyomaviruses; Epstein-Barr viruses; papilloma viruses; herpes virus;vaccinia virus; polio virus; and RNA virus such as a retrovirus. One canreadily employ other vectors well-known in the art. Certain viralvectors are based on non-cytopathic eukaryotic viruses in whichnon-essential genes have been replaced with the gene of interest.Non-cytopathic viruses include retroviruses, the life cycle of whichinvolves reverse transcription of genomic viral RNA into DNA withsubsequent proviral integration into host cellular DNA. Retroviruseshave been approved for human gene therapy trials. Most useful are thoseretroviruses that are replication-deficient (i.e., capable of directingsynthesis of the desired proteins, but incapable of manufacturing aninfectious particle). Such genetically altered retroviral expressionvectors have general utility for the high-efficiency transduction ofgenes in vivo. Standard protocols for producing replication-deficientretroviruses (including the steps of incorporation of exogenous geneticmaterial into a plasmid, transfection of a packaging cell line withplasmid, production of recombinant retroviruses by the packaging cellline, collection of viral particles from tissue culture media, andinfection of the target cells with viral particles) are provided inKriegler, M., Gene Transfer and Expression, A Laboratory Manual, W.H.Freeman Co., New York (1990) and Murry, E. J., Methods in MolecularBiology, Vol. 7, Humana Press, Inc., Cliffton, N.J. (1991).

In one embodiment, the virus is an adeno-associated virus, adouble-stranded DNA virus. The adeno-associated virus can be engineeredto be replication-deficient and is capable of infecting a wide range ofcell types and species. It further has advantages such as heat and lipidsolvent stability; high transduction frequencies in cells of diverselineages, including hemopoietic cells; and lack of superinfectioninhibition thus allowing multiple series of transductions. Reportedly,the adeno-associated virus can integrate into human cellular DNA in asite-specific manner, thereby minimizing the possibility of insertionalmutagenesis and variability of inserted gene expression characteristicof retroviral infection. In addition, wild-type adeno-associated virusinfections have been followed in tissue culture for greater than 100passages in the absence of selective pressure, implying that theadeno-associated virus genomic integration is a relatively stable event.The adeno-associated virus can also function in an extrachromosomalfashion.

Other vectors include plasmid vectors. Plasmid vectors have beenextensively described in the art and are well-known to those of skill inthe art. See, e.g., Sambrook et al., Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. Inthe last few years, plasmid vectors have been found to be particularlyadvantageous for delivering genes to cells in vivo because of theirinability to replicate within and integrate into a host genome. Theseplasmids, however, having a promoter compatible with the host cell, canexpress a peptide from a gene operably encoded within the plasmid. Somecommonly used plasmids available from commercial suppliers includepBR322, pUC18, pUC19, various pcDNA plasmids, pRC/CMV, various pCMVplasmids, pSV40, and pBlueScript. Additional examples of specificplasmids include pcDNA3.1, catalog number V79020; pcDNA3.1/hygro,catalog number V87020; pcDNA4/myc-His, catalog number V86320; andpBudCE4.1, catalog number V53220, all from Invitrogen (Carlsbad,Calif.). Other plasmids are well-known to those of ordinary skill in theart. Additionally, plasmids may be custom designed using standardmolecular biology techniques to remove and/or add specific fragments ofDNA.

In one insect expression system that may be used to produce the proteinsof the invention, Autographa californica nuclear polyhidrosis virus(AcNPV) is used as a vector to express the foreign genes. The virusgrows in Spodoptera frugiperda cells. A coding sequence may be clonedinto non-essential regions (for example, the polyhedron gene) of thevirus and placed under control of an ACNPV promoter (for example, thepolyhedron promoter). Successful insertion of a coding sequence willresult in inactivation of the polyhedron gene and production ofnon-occluded recombinant virus (i.e., virus lacking the proteinaceouscoat coded for by the polyhedron gene). These recombinant viruses arethen used to infect Spodoptera frugiperda cells in which the insertedgene is expressed. (see, e.g., Smith et al. (1983) J Virol 46:584; U.S.Pat. No. 4,215,051). Further examples of this expression system may befound in Ausubel et al., eds. (1989) Current Protocols in MolecularBiology, Vol. 2, Greene Publish. Assoc. & Wiley Interscience.

Another system which can be used to express the proteins of theinvention is the glutamine synthetase gene expression system, alsoreferred to as the “GS expression system” (Lonza Biologics PLC,Berkshire UK). This expression system is described in detail in U.S.Pat. No. 5,981,216.

In mammalian host cells, a number of viral based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, a coding sequence may be ligated to an adenovirustranscription/translation control complex, e.g., the late promoter andtripartite leader sequence. This chimeric gene may then be inserted inthe adenovirus genome by in vitro or in vivo recombination. Insertion ina non-essential region of the viral genome (e.g., region E1 or E3) willresult in a recombinant virus that is viable and capable of expressingpeptide in infected hosts. See, e.g., Logan & Shenk (1984) Proc NatlAcad Sci USA 81:3655). Alternatively, the vaccinia 7.5 K promoter may beused. See, e.g., Mackett et al. (1982) Proc Natl Acad Sci USA 79:7415;Mackett et al. (1984) J Virol 49:857; Panicali et al. (1982) Proc NatlAcad Sci USA 79:4927.

To increase efficiency of production, the polynucleotides can bedesigned to encode multiple units of the protein of the inventionseparated by enzymatic cleavage sites. The resulting polypeptide can becleaved (e.g., by treatment with the appropriate enzyme) in order torecover the polypeptide units. This can increase the yield ofpolypeptides driven by a single promoter. When used in appropriate viralexpression systems, the translation of each polypeptide encoded by themRNA is directed internally in the transcript; e.g., by an internalribosome entry site, IRES. Thus, the polycistronic construct directs thetranscription of a single, large polycistronic mRNA which, in turn,directs the translation of multiple, individual polypeptides. Thisapproach eliminates the production and enzymatic processing ofpolyproteins and may significantly increase the yield of polypeptidesdriven by a single promoter.

Vectors used in transformation will usually contain a selectable markerused to identify transformants. In bacterial systems, this can includean antibiotic resistance gene such as ampicillin or kanamycin.Selectable markers for use in cultured mammalian cells include genesthat confer resistance to drugs, such as neomycin, hygromycin, andmethotrexate. The selectable marker may be an amplifiable selectablemarker. One amplifiable selectable marker is the dihydrofolate reductase(DHFR) gene. Simonsen C C et al. (1983) Proc Natl Acad Sci USA80:2495-9. Selectable markers are reviewed by Thilly (1986) MammalianCell Technology, Butterworth Publishers, Stoneham, Mass., and the choiceof selectable markers is well within the level of ordinary skill in theart.

Selectable markers may be introduced into the cell on a separate plasmidat the same time as the gene of interest, or they may be introduced onthe same plasmid. If on the same plasmid, the selectable marker and thegene of interest may be under the control of different promoters or thesame promoter, the latter arrangement producing a dicistronic message.Constructs of this type are known in the art (for example, U.S. Pat. No.4,713,339).

The expression vectors can encode for tags that permit easy purificationof the recombinantly produced protein. Examples include, but are notlimited to, vector pUR278 (Ruther et al. (1983) EMBO J 2:1791), in whichcoding sequences for the protein to be expressed may be ligated into thevector in frame with the lac z coding region so that a tagged fusionprotein is produced; pGEX vectors may be used to express proteins of theinvention with a glutathione S-transferase (GST) tag. These proteins areusually soluble and can easily be purified from cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. The vectors include cleavage sites (thrombin or Factor Xaprotease or PRESCISSION PROTEASE™ (Pharmacia, Peapack, N.J.)) for easyremoval of the tag after purification.

The expression vector or vectors are then transfected or co-transfectedinto a suitable target cell, which will express the polypeptides.Transfection techniques known in the art include, but are not limitedto, calcium phosphate precipitation (Wigler et al. (1978) Cell 14:725),electroporation (Neumann et al. (1982) EMBO J 1:841), and liposome-basedreagents. A variety of host-expression vector systems may be utilized toexpress the proteins described herein including both prokaryotic andeukaryotic cells. These include, but are not limited to, microorganismssuch as bacteria (e.g., E. coli) transformed with recombinantbacteriophage DNA or plasmid DNA expression vectors containing anappropriate coding sequence; yeast or filamentous fungi transformed withrecombinant yeast or fungi expression vectors containing an appropriatecoding sequence; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing an appropriate codingsequence; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus or tobacco mosaic virus) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing an appropriate coding sequence; or animal cellsystems, including mammalian cells (e.g., HEK 293, CHO, Cos, HeLa,HKB11, and BHK cells).

In one embodiment, the host cell is a eukaryotic cell. As used herein, aeukaryotic cell refers to any animal or plant cell having a definitivenucleus. Eukaryotic cells of animals include cells of vertebrates, e.g.,mammals, and cells of invertebrates, e.g., insects. Eukaryotic cells ofplants specifically can include, without limitation, yeast cells. Aeukaryotic cell is distinct from a prokaryotic cell, e.g., bacteria.

In certain embodiments, the eukaryotic cell is a mammalian cell. Amammalian cell is any cell derived from a mammal. Mammalian cellsspecifically include, but are not limited to, mammalian cell lines. Inone embodiment, the mammalian cell is a human cell. In anotherembodiment, the mammalian cell is a HEK 293 cell, which is a humanembryonic kidney cell line. HEK 293 cells are available as CRL-1533 fromAmerican Type Culture Collection, Manassas, Va., and as 293-H cells,Catalog No. 11631-017 or 293-F cells, Catalog No. 11625-019 fromInvitrogen (Carlsbad, Calif.). In some embodiments, the mammalian cellis a PER.C6® cell, which is a human cell line derived from retina.PER.C6® cells are available from Crucell (Leiden, The Netherlands). Inother embodiments, the mammalian cell is a Chinese hamster ovary (CHO)cell. CHO cells are available from American Type Culture Collection,Manassas, Va. (e.g., CHO-K1; CCL-61). In still other embodiments, themammalian cell is a baby hamster kidney (BHK) cell. BHK cells areavailable from American Type Culture Collection, Manassas, Va. (e.g.,CRL-1632). In some embodiments, the mammalian cell is a HKB11 cell,which is a hybrid cell line of a HEK293 cell and a human B cell line.Mei et al., Mol. Biotechnol. 34(2): 165-78 (2006).

In one embodiment, a plasmid encoding the VWF fragment or the chimericprotein of the invention further includes a selectable marker, e.g.,zeocin resistance, and is transfected into HEK 293 cells, for productionof the VWF fragment or the chimeric protein.

In another embodiment, a first plasmid comprising a Factor VIII-Fcfusion coding sequence and a first selectable marker, e.g., a zeocinresistance gene, and a second plasmid comprising a VWF fragment-Fccoding sequence and a second selectable marker, e.g., a neomycinresistance gene, are cotransfected into HEK 293 cells, for production ofFactor VIII-Fc and VWF-Fc hybrid. The first and second plasmids can beintroduced in equal amounts (i.e., 1:1 ratio), or they can be introducedin unequal amounts.

In some embodiments, a first plasmid including a Factor VIII-Fc fusioncoding sequence and a first selectable marker, e.g., a zeocin resistancegene, and a second plasmid including a VWF fragment-Fc coding sequenceand a second selectable marker, e.g., a neomycin resistance gene, and athird plasmid including a protein convertase coding sequence (e.g., PC5or Furin) and a third selectable marker, e.g., a hygromycin resistancegene, are cotransfected into HEK 293 cells, for production of FactorVIII-VWF fragment hybrid. The first and second plasmids can beintroduced in equal amounts (i.e., 1:1 molar ratio), or they can beintroduced in unequal amounts. In certain embodiments, a first plasmid,including a Factor VIII-Fc fusion coding sequence, a VWF fragment-Fccoding sequence, and a first selectable marker, e.g., a zeocinresistance gene, and a second plasmid including a protein convertasecoding sequence (e.g., PC5 or Furin) and a second selectable marker,e.g., a hygromycin resistance gene, are cotransfected into HEK 293cells, for production of Factor VIII-VWF-fragment hybrid. In oneembodiment, the nucleotide sequence encoding the FVIII-Fc sequence andthe VWF fragment-Fc sequence can be connected to encode one singlepolypeptide. In another embodiment, the nucleotide sequence encoding theFVIII-Fc sequence and the VWF fragment-Fc sequence can be encoded as twopolypeptide chains. The promoters for the Factor VIII-Fc fusion codingsequence and the VWF fragment-Fc coding sequence can be different orthey can be the same.

In some embodiments, a plasmid comprising Furin is co-transfected withthe plasmid containing the Factor VIII-Fc coding sequence and/or VWFfragment-Fc coding sequence. In some embodiments, the Furin protein ison the same plasmid comprising the Factor VIII-Fc fusion codingsequence. In some embodiments, the Furin protein is on the same plasmidcomprising the VWF fragment-Fc coding sequence. In some embodiments, theFurin protein is on a separate plasmid.

In still other embodiments, transfected cells are stably transfected.These cells can be selected and maintained as a stable cell line, usingconventional techniques known to those of skill in the art.

Host cells containing DNA constructs of the protein are grown in anappropriate growth medium. As used herein, the term “appropriate growthmedium” means a medium containing nutrients required for the growth ofcells. Nutrients required for cell growth may include a carbon source, anitrogen source, essential amino acids, vitamins, minerals, and growthfactors. Optionally, the media can contain one or more selectionfactors. Optionally the media can contain bovine calf serum or fetalcalf serum (FCS). In one embodiment, the media contains substantially noIgG. The growth medium will generally select for cells containing theDNA construct by, for example, drug selection or deficiency in anessential nutrient which is complemented by the selectable marker on theDNA construct or co-transfected with the DNA construct. Culturedmammalian cells are generally grown in commercially availableserum-containing or serum-free media (e.g., MEM, DMEM, DMEM/F12). In oneembodiment, the medium is CD293 (Invitrogen, Carlsbad, Calif.). Inanother embodiment, the medium is CD17 (Invitrogen, Carlsbad, Calif.).Selection of a medium appropriate for the particular cell line used iswithin the level of those ordinary skilled in the art.

In order to co-express the VWF fragment and a second heterologous moietyor a FVIII protein, the host cells are cultured under conditions thatallow expression of both the VWF fragment and a second heterologousmoiety or a FVIII protein. As used herein, culturing refers tomaintaining living cells in vitro for at least a definite time.Maintaining can, but need not include, an increase in population ofliving cells. For example, cells maintained in culture can be static inpopulation, but still viable and capable of producing a desired product,e.g., a recombinant protein or recombinant fusion protein. Suitableconditions for culturing eukaryotic cells are well known in the art andinclude appropriate selection of culture media, media supplements,temperature, pH, oxygen saturation, and the like. For commercialpurposes, culturing can include the use of any of various types ofscale-up systems including shaker flasks, roller bottles, hollow fiberbioreactors, stirred-tank bioreactors, airlift bioreactors, Wavebioreactors, and others.

The cell culture conditions are also selected to allow association ofthe VWF fragment with the second heterologous moiety or a FVIII protein.Conditions that allow expression of the VWF fragment and/or the FVIIIprotein, may include the presence of a source of vitamin K. For example,in one embodiment, stably transfected HEK 293 cells are cultured inCD293 media (Invitrogen, Carlsbad, Calif.) or OptiCHO media (Invitrogen,Carlsbad, Calif.) supplemented with 4 mM glutamine.

In one aspect, the present invention is directed to a method ofexpressing, making, or producing the VWF fragment of the inventioncomprising a) transfecting a host cell with a polynucleotide encodingthe VWF fragment and b) culturing the host cell in a culture mediumunder a condition suitable for expressing the VWF fragment, wherein theVWF fragment is expressed. In one embodiment, the invention is drawn toa method of producing a mature VWF protein or a fragment thereofcomprising a) transfecting a host cell with a first polynucleotideencoding the VWF protein or a fragment thereof, which is fused to apropeptide of VWF, and a second polynucleotide encoding a proteinconvertase, e.g., PC5, PC7, or Furin and b) culturing the host cell in aculture medium under a condition suitable for expressing the mature VWFprotein or fragment thereof. The polynucleotide encoding the VWF proteinor a fragment thereof can also be fused to a prepeptide of VWF. Theprepeptide sequence can be cleaved during insertion to the endoplasmicreticulum before secretion.

In another aspect, the invention is directed to a method of expressing,making, or producing a chimeric protein comprising the VWF fragmentlinked to or associated with a heterologous moiety or a FVIII proteincomprising a) transfecting one or more host cells with a polynucleotideor a set of polynucleotides encoding the chimeric protein and b)culturing the host cell in a culture medium under conditions suitablefor expressing the chimeric protein. In one embodiment, the invention isdrawn to a method of expressing, making, or producing a chimeric proteincomprising a) transfecting a host cell with a first polynucleotideencoding a VWF fragment linked to a heterologous moiety and a secondpolynucleotide encoding a FVIII protein linked to a heterologous moietyand b) culturing the host cell in a culture medium under conditionssuitable for expressing the chimeric protein. The first polynucleotideand the second polynucleotide can be in one vector or two vectors. Inanother embodiment, the invention is drawn to a method of expressing,making, or producing a chimeric protein comprising a) transfecting ahost cell with a first polynucleotide encoding a VWF fragment linked toa heterologous moiety, a second polynucleotide encoding a FVIII proteinlinked to a heterologous moiety, and a third polynucleotide encoding aprotein convertase, and b) culturing the host cell in a culture mediumunder conditions suitable for expressing the chimeric protein. In otherembodiments, the invention is drawn to a method of expressing, making,or producing a chimeric protein comprising a) transfecting a host cellwith a first polynucleotide encoding a VWF fragment comprising a D′domain and a D3 domain linked to a heterologous moiety, a secondpolynucleotide encoding a FVIII protein linked to a heterologous moiety,and a third polynucleotide encoding a D1 domain and a D2 domain of VWF,and b) culturing the host cell in a culture medium under conditionssuitable for expressing the chimeric protein. In one embodiment, thefirst polynucleotide, the second polynucleotide, and the thirdpolynucleotide can be in one vector or separate vectors. In anotherembodiment, the first polynucleotide and the second polynucleotide canbe in one vector, and the third polynucleotide can be another vector. Inother embodiments, the first polynucleotide and the third polynucleotidecan be in one vector, and the second polynucleotide can be anothervector. In some embodiments, the second polynucleotide and the thirdpolynucleotide can be in one vector and the first polynucleotide can bein another vector.

In further embodiments, the protein product containing the VWF fragmentor the chimeric protein comprising the VWF fragment is secreted into themedia. Media is separated from the cells, concentrated, filtered, andthen passed over two or three affinity columns, e.g., a protein A columnand one or two anion exchange columns.

In certain aspects, the present invention relates to the VWF fragment orthe chimeric polypeptide produced by the methods described herein.

In vitro production allows scale-up to give large amounts of the desiredaltered polypeptides of the invention. Techniques for mammalian cellcultivation under tissue culture conditions are known in the art andinclude homogeneous suspension culture, e.g. in an airlift reactor or ina continuous stirrer reactor, or immobilized or entrapped cell culture,e.g. in hollow fibers, microcapsules, on agarose microbeads or ceramiccartridges. If necessary and/or desired, the solutions of polypeptidescan be purified by the customary chromatography methods, for example gelfiltration, ion-exchange chromatography, hydrophobic interactionchromatography (HIC, chromatography over DEAE-cellulose or affinitychromatography.

Pharmaceutical Composition

Compositions containing the VWF fragment or the chimeric protein of thepresent invention may contain a suitable pharmaceutically acceptablecarrier. For example, they may contain excipients and/or auxiliariesthat facilitate processing of the active compounds into preparationsdesigned for delivery to the site of action.

The pharmaceutical composition can be formulated for parenteraladministration (i.e. intravenous, subcutaneous, or intramuscular) bybolus injection. Formulations for injection can be presented in unitdosage form, e.g., in ampoules or in multidose containers with an addedpreservative. The compositions can take such forms as suspensions,solutions, or emulsions in oily or aqueous vehicles, and containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the active ingredient can be in powder form forconstitution with a suitable vehicle, e.g., pyrogen free water.

Suitable formulations for parenteral administration also include aqueoussolutions of the active compounds in water-soluble form, for example,water-soluble salts. In addition, suspensions of the active compounds asappropriate oily injection suspensions may be administered. Suitablelipophilic solvents or vehicles include fatty oils, for example, sesameoil, or synthetic fatty acid esters, for example, ethyl oleate ortriglycerides. Aqueous injection suspensions may contain substances,which increase the viscosity of the suspension, including, for example,sodium carboxymethyl cellulose, sorbitol and dextran. Optionally, thesuspension may also contain stabilizers. Liposomes also can be used toencapsulate the molecules of the invention for delivery into cells orinterstitial spaces. Exemplary pharmaceutically acceptable carriers arephysiologically compatible solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, water, saline, phosphate buffered saline, dextrose, glycerol,ethanol and the like. In some embodiments, the composition comprisesisotonic agents, for example, sugars, polyalcohols such as mannitol,sorbitol, or sodium chloride. In other embodiments, the compositionscomprise pharmaceutically acceptable substances such as wetting agentsor minor amounts of auxiliary substances such as wetting or emulsifyingagents, preservatives or buffers, which enhance the shelf life oreffectiveness of the active ingredients.

Compositions of the invention may be in a variety of forms, including,for example, liquid (e.g., injectable and infusible solutions),dispersions, suspensions, semi-solid and solid dosage forms. Thepreferred form depends on the mode of administration and therapeuticapplication.

The composition can be formulated as a solution, micro emulsion,dispersion, liposome, or other ordered structure suitable to high drugconcentration. Sterile injectable solutions can be prepared byincorporating the active ingredient in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedabove, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the active ingredient into asterile vehicle that contains a basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-dryingthat yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution. Theproper fluidity of a solution can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prolonged absorption of injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

The active ingredient can be formulated with a controlled-releaseformulation or device. Examples of such formulations and devices includeimplants, transdermal patches, and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, for example, ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Methods for the preparation ofsuch formulations and devices are known in the art. See e.g., Sustainedand Controlled Release Drug Delivery Systems, J. R. Robinson, ed.,Marcel Dekker, Inc., New York, 1978.

Injectable depot formulations can be made by forming microencapsulatedmatrices of the drug in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the polymer employed, the rate of drug release can becontrolled. Other exemplary biodegradable polymers are polyorthoestersand polyanhydrides. Depot injectable formulations also can be preparedby entrapping the drug in liposomes or microemulsions.

Supplementary active compounds can be incorporated into thecompositions. In one embodiment, the VWF fragment or the chimericprotein of the invention is formulated with another clotting factor, ora variant, fragment, analogue, or derivative thereof. For example, theclotting factor includes, but is not limited to, factor V, factor VII,factor VIII, factor IX, factor X, factor XI, factor XII, factor XIII,prothrombin, fibrinogen, von Willebrand factor or recombinant solubletissue factor (rsTF) or activated forms of any of the preceding. Theclotting factor of hemostatic agent can also include anti-fibrinolyticdrugs, e.g., epsilon-amino-caproic acid, tranexamic acid.

Dosage regimens may be adjusted to provide the optimum desired response.For example, a single bolus may be administered, several divided dosesmay be administered over time, or the dose may be proportionally reducedor increased as indicated by the exigencies of the therapeuticsituation. It is advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.See, e.g., Remington's Pharmaceutical Sciences (Mack Pub. Co., Easton,Pa. 1980).

In addition to the active compound, the liquid dosage form may containinert ingredients such as water, ethyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, dimethylformamide, oils, glycerol, tetrahydrofurfuryl alcohol,polyethylene glycols, and fatty acid esters of sorbitan.

Non-limiting examples of suitable pharmaceutical carriers are alsodescribed in Remington's Pharmaceutical Sciences by E. W. Martin. Someexamples of excipients include starch, glucose, lactose, sucrose,gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerolmonostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol, and the like. The composition canalso contain pH buffering reagents, and wetting or emulsifying agents.

For oral administration, the pharmaceutical composition can take theform of tablets or capsules prepared by conventional means. Thecomposition can also be prepared as a liquid for example a syrup or asuspension. The liquid can include suspending agents (e.g., sorbitolsyrup, cellulose derivatives or hydrogenated edible fats), emulsifyingagents (lecithin or acacia), non-aqueous vehicles (e.g., almond oil,oily esters, ethyl alcohol, or fractionated vegetable oils), andpreservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbicacid). The preparations can also include flavoring, coloring andsweetening agents. Alternatively, the composition can be presented as adry product for constitution with water or another suitable vehicle.

For buccal administration, the composition may take the form of tabletsor lozenges according to conventional protocols.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of a nebulizedaerosol with or without excipients or in the form of an aerosol sprayfrom a pressurized pack or nebulizer, with optionally a propellant,e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoromethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit can be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof, e.g., gelatin for use in an inhaler or insufflator can be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

The pharmaceutical composition can also be formulated for rectaladministration as a suppository or retention enema, e.g., containingconventional suppository bases such as cocoa butter or other glycerides.

Gene Therapy

A VWF fragment or chimeric protein thereof of the invention can beproduced in vivo in a mammal, e.g., a human patient, using a genetherapy approach to treatment of a bleeding disease or disorder selectedfrom the group consisting of a bleeding coagulation disorder,hemarthrosis, muscle bleed, oral bleed, hemorrhage, hemorrhage intomuscles, oral hemorrhage, trauma, trauma capitis, gastrointestinalbleeding, intracranial hemorrhage, intra-abdominal hemorrhage,intrathoracic hemorrhage, bone fracture, central nervous systembleeding, bleeding in the retropharyngeal space, bleeding in theretroperitoneal space, and bleeding in the illiopsoas sheath would betherapeutically beneficial. In one embodiment, the bleeding disease ordisorder is hemophilia. In another embodiment, the bleeding disease ordisorder is hemophilia A. This involves administration of a suitable VWFfragment or chimeric protein-encoding nucleic acid operably linked tosuitable expression control sequences. In certain embodiment, thesesequences are incorporated into a viral vector. Suitable viral vectorsfor such gene therapy include adenoviral vectors, lentiviral vectors,baculoviral vectors, Epstein Barr viral vectors, papovaviral vectors,vaccinia viral vectors, herpes simplex viral vectors, and adenoassociated virus (AAV) vectors. The viral vector can be areplication-defective viral vector. In other embodiments, a adenoviralvector has a deletion in its E1 gene or E3 gene. When an adenoviralvector is used, the mammal may not be exposed to a nucleic acid encodinga selectable marker gene. In other embodiments, the sequences areincorporated into a non-viral vector known to those skilled in the art.

Methods of Using VWF Fragment or Chimeric Protein

One aspect of the present invention is directed to preventing orinhibiting FVIII interaction with endogenous VWF by blocking orshielding the VWF binding site on the FVIII from endogenous VWF. In oneembodiment, the invention is directed to a method of constructing aFVIII protein having half-life longer than wild-type FVIII or a FVIIImonomer-dimer hybrid, the method comprising covalently associating anadjunct moiety with the FVIII protein, thereby making a chimeric proteincomprising the FVIII protein and the adjunct moiety, wherein the adjunctmoiety shields or prevents the FVIII protein interaction with endogenousVWF. The chimeric protein useful in the method includes any one or morechimeric protein described herein.

Another aspect of the invention includes a method of administering to asubject in need thereof a FVIII protein having half-life longer thanwild-type FVIII or a FVIII monomer-dimer hybrid, which consists of twopolypeptide chains, a first chain consisting of an amino acid sequenceencoding FVIII and an Fc region and a second chain consisting of an Fcregion, wherein the method comprises administering the VWF fragmentdescribed herein or the chimeric protein described herein to thesubject. The FVIII amino acid sequence in the monomer-dimer hybrid canbe SQ FVIII or wild-type FVIII.

In one embodiment, the invention is directed to a method of using anadjunct moiety, e.g., a VWF fragment described herein or a chimericprotein comprising the VWF fragment, to prevent or inhibit endogenousVWF interaction with a FVIII protein. In another embodiment, a FVIIIprotein that is capable of interacting with the VWF fragment isendogenous FVIII. In other embodiments, a FVIII protein that is capableof interacting with the VWF fragment is a FVIII composition separatelyadministered to a subject before or after or simultaneously with the VWFfragment or the chimeric protein comprising the VWF fragment. In otherembodiments, a FVIII protein that is capable of binding to the VWFfragment is a FVIII composition administered to a subject together withthe VWF fragment or the chimeric protein. In still other embodiments, aFVIII protein that is capable of binding to the VWF fragment is FVIIIpresent with the VWF fragment or associated with the VWF fragment in thechimeric protein. The VWF fragment or the chimeric protein comprisingthe VWF fragment binds to, or is associated with, the FVIII protein andthus extends the half-life of the FVIII protein bound to the VWFfragment or the chimeric protein. The FVIII protein bound to the VWFfragment or the chimeric protein is shielded or protected from theclearance pathway of VWF and thus has reduced clearance compared to theFVIII protein not bound to the VWF fragment or the chimeric protein. Theshielded FVIII protein thus has a longer half-life than a FVIII proteinnot bound to or associated with the VWF fragment or the chimericprotein. In certain embodiments, the FVIII protein associated with orprotected by a VWF fragment or a chimeric protein of the invention isnot cleared by a VWF clearance receptor. In other embodiments, the FVIIIprotein associated with or protected by a VWF fragment or a chimericprotein is cleared from the system slower than the FVIII protein that isnot associated with or protected by the VWF fragment.

In one aspect, the VWF fragment of this invention or the chimericprotein comprising the same has reduced clearance from circulation asthe VWF fragment or the chimeric protein does not contain a VWFclearance receptor binding site. The VWF fragment prevents or inhibitsclearance of FVIII bound to or associated with the VWF fragment from thesystem through the VWF clearance pathway. The VWF fragments useful forthe present invention can also provide at least one or more VWF-likeFVIII protection properties that are provided by endogenous VWF. Incertain embodiments, the VWF fragments can also mask one or more FVIIIclearance receptor binding site, thereby preventing clearance of FVIIIby its own clearance pathway.

In another aspect, the VWF fragment or chimeric protein of the inventioncan be used to treat or prevent a disease or disorder associated with aType 2N von Willebrand disease (VWD). Type 2N VWD is a qualitative VWFdefect resulting from defective VWF binding to FVIII and consequentlyresulting in low levels of circulating FVIII. Therefore, the VWFfragment or chimeric protein of the invention by binding to or beingbound to the FVIII protein not only stabilizes the FVIII protein, butalso prevents clearance of the FVIII protein from the circulation.

In some embodiments, the prevention or inhibition of a FVIII proteinbinding to endogenous VWF by the VWF fragment or chimeric protein can bein vitro or in vivo.

Also provided is a method of increasing the half-life of a FVIII proteincomprising administering the VWF fragment or the chimeric proteincomprising the VWF fragment and a FVIII protein to a subject in needthereof. The half-life of non-activated FVIII bound to or associatedwith full-length VWF is about 12 to 14 hours in plasma. In VWD type 3,wherein there is almost no VWF in circulation, the half-life of FVIII isonly about six hours, leading to symptoms of mild to moderate hemophiliaA in such patients due to decreased concentrations of FVIII. Thehalf-life of the FVIII protein linked to or associated with the VWFfragment of the present invention can increase at least about 1.5 times,1.6 times, 1.7 times, 1.8 times, 1.9 times, 2.0 times, 2.1 times, 2.2times, 2.3 times, 2.4 times, 2.6 times, 2.7. times, 2.8 times, 2.9times, 3.0 times, 3.1 times, 3.2 times, 3.3 times, 3.4 times, 3.5 times,3.6 times, 3.7 times, 3.8 times, 3.9 times, or 4.0 times higher than thehalf-life of the non-activated FVIII bound to or associated withfull-length VWF. In one embodiment, the half-life of the FVIII proteinlinked to or associated with the VWF fragment in the chimeric proteinincreases at least about 2 times, 2.5 times, 3.0 times, 3.5 times, 4.0times, 4.5 times, 5.0 times, 5.5 times, 6.0 times, 7 times, 8 times, 9times, or 10 times higher than the half-life of the non-activated FVIIIbound to or associated with full-length VWF. In another embodiment, thehalf-life of the FVIII protein linked to or associated with the VWFfragment in the chimeric protein increases about 2 to about 5 times,about 3 to about 10 times, about 5 to about 15 times, about 10 to about20 times, about 15 to about 25 times, about 20 to about 30 times, about25 to about 35 times, about 30 to about 40 times, about 35 to about 45times higher than the half-life of the non-activated FVIII bound to orassociated with full-length VWF. In a specific embodiment, the half-lifeof the FVIII protein linked to or associated with the VWF fragment inthe chimeric protein increases at least about 30, 31, 32, 33, 34, 35,36, 37, 38, 39, or 40 times higher than the half-life of the wild typeFVIII in a FVIII and VWF double knockout mouse. In some embodiments, thehalf-life of the chimeric protein comprising the VWF fragment fused to afirst heterologous moiety, e.g., a first Fc region, and a FVIII proteinlinked to a second heterologous moiety, e.g., a second Fc region islonger than the half-life of a chimeric protein comprising a FVIIIprotein and two Fc regions, wherein the FVIII protein is linked to oneof the two Fc regions (i.e., FVIII monomer-dimer hybrid). In otherembodiments, the half-life of the chimeric protein comprising the VWFfragment fused to a first heterologous moiety, e.g., a first Fc region,and a FVIII protein linked to a second heterologous moiety, e.g., asecond Fc region is at least about 1.5 times, 2 times, 2.5 times, 3.5times, 3.6 times, 3.7 times, 3.8 times, 3.9 times, 4.0 times, 4.5 times,or 5.0 times the half-life of a chimeric protein comprising a FVIIIprotein and two Fc regions, wherein the FVIII protein is linked to oneof the two Fc regions (i.e., FVIII monomer-dimer hybrid).

In some embodiments, as a result of the invention the half-life of theFVIII protein is extended compared to a FVIII protein without the VWFfragment or wildtype FVIII. The half-life of the FVIII protein is atleast about 1.5 times, at least about 2 times, at least about 2.5 times,at least about 3 times, at least about 4 times, at least about 5 times,at least about 6 times, at least about 7 times, at least about 8 times,at least about 9 times, at least about 10 times, at least about 11times, or at least about 12 times longer than the half-life of a FVIIIprotein without the VWF fragment. In one embodiment, the half-life ofFVIII is about 1.5-fold to about 20-fold, about 1.5 fold to about 15fold, or about 1.5 fold to about 10 fold longer than the half-life ofwild-type FVIII. In another embodiment, the half-life of the FVIII isextended about 2-fold to about 10-fold, about 2-fold to about 9-fold,about 2-fold to about 8-fold, about 2-fold to about 7-fold, about 2-foldto about 6-fold, about 2-fold to about 5-fold, about 2-fold to about4-fold, about 2-fold to about 3-fold, about 2.5-fold to about 10-fold,about 2.5-fold to about 9-fold, about 2.5-fold to about 8-fold, about2.5-fold to about 7-fold, about 2.5-fold to about 6-fold, about 2.5-foldto about 5-fold, about 2.5-fold to about 4-fold, about 2.5-fold to about3-fold, about 3-fold to about 10-fold, about 3-fold to about 9-fold,about 3-fold to about 8-fold, about 3-fold to about 7-fold, about 3-foldto about 6-fold, about 3-fold to about 5-fold, about 3-fold to about4-fold, about 4-fold to about 6 fold, about 5-fold to about 7-fold, orabout 6-fold to about 8 fold as compared to wild-type FVIII or a FVIIIprotein without the VWF fragment. In other embodiments, the half-life ofFVIII is at least about 17 hours, at least about 18 hours, at leastabout 19 hours, at least about 20 hours, at least about 21 hours, atleast about 22 hours, at least about 23 hours, at least about 24 hours,at least about 25 hours, at least about 26 hours, at least about 27hours, at least about 28 hours, at least about 29 hours, at least about30 hours, at least about 31 hours, at least about 32 hours, at leastabout 33 hours, at least about 34 hours, at least about 35 hours, atleast about 36 hours, at least about 48 hours, at least about 60 hours,at least about 72 hours, at least about 84 hours, at least about 96hours, or at least about 108 hours. In still other embodiments, thehalf-life of FVIII is about 15 hours to about two weeks, about 16 hoursto about one week, about 17 hours to about one week, about 18 hours toabout one week, about 19 hours to about one week, about 20 hours toabout one week, about 21 hours to about one week, about 22 hours toabout one week, about 23 hours to about one week, about 24 hours toabout one week, about 36 hours to about one week, about 48 hours toabout one week, about 60 hours to about one week, about 24 hours toabout six days, about 24 hours to about five days, about 24 hours toabout four days, about 24 hours to about three days, or about 24 hoursto about two days.

In some embodiments, the average half-life of the FVIII protein persubject is about 15 hours, about 16 hours, about 17 hours, about 18hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours,about 23 hours, about 24 hours (1 day), about 25 hours, about 26 hours,about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours,about 36 hours, about 40 hours, about 44 hours, about 48 hours (2 days),about 54 hours, about 60 hours, about 72 hours (3 days), about 84 hours,about 96 hours (4 days), about 108 hours, about 120 hours (5 days),about six days, about seven days (one week), about eight days, aboutnine days, about 10 days, about 11 days, about 12 days, about 13 days,or about 14 days.

In a specific embodiment, a half-life of the chimeric protein of theinvention is about two fold longer than the half-life of wild-type FVIIIor BDD FVIII. In another embodiment, a half-life of the chimeric proteinis about three fold longer than the half-life of wild-type FVIII or BDDFVIII.

In addition, the invention provides a method of treating or preventing ableeding disease or disorder comprising administering an effectiveamount of the VWF fragment or the chimeric protein (e.g., a chimericprotein comprising the VWF fragment linked to a first heterologousmoiety, e.g., a first Fc region, and a FVIII protein linked to a secondheterologous moiety, e.g., a second Fc region, wherein the VWF fragmentis bound to or associated with the FVIII protein). In one embodiment,the bleeding disease or disorder is selected from the group consistingof a bleeding coagulation disorder, hemarthrosis, muscle bleed, oralbleed, hemorrhage, hemorrhage into muscles, oral hemorrhage, trauma,trauma capitis, gastrointestinal bleeding, intracranial hemorrhage,intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture,central nervous system bleeding, bleeding in the retropharyngeal space,bleeding in the retroperitoneal space, and bleeding in the illiopsoassheath. In a specific embodiment, the bleeding disease or disorder ishemophilia A.

The VWF fragment and the chimeric protein comprising an adjunct moiety,e.g., the VWF fragment described herein and a FVIII protein prepared bythe invention has many uses as will be recognized by one skilled in theart, including, but not limited to methods of treating a subject havinga hemostatic disorder and methods of treating a subject in need of ageneral hemostatic agent. In one embodiment, the invention relates to amethod of treating a subject having a hemostatic disorder comprisingadministering a therapeutically effective amount of the VWF fragment orthe chimeric protein.

The FVIII protein portion in the chimeric protein treats or prevents ahemostatic disorder by serving as a cofactor to Factor IX on anegatively charged phospholipid surface, thereby forming a Xase complex.The binding of activated coagulation factors to a phospholipid surfacelocalizes this process to sites of vascular damage. On a phospholipidsurface, Factor Villa increases the maximum velocity of Factor Xactivation by Factor IXa, by approximately 200,000-fold, leading to thelarge second burst of thrombin generation.

The chimeric protein comprising an adjunct moiety, e.g., a VWF fragment,and a FVIII protein can be used to treat any hemostatic disorder. Thehemostatic disorders that may be treated by administration of thechimeric protein of the invention include, but are not limited to,hemophilia A, as well as deficiencies or structural abnormalitiesrelating to Factor VIII. In one embodiment, the hemostatic disorder ishemophilia A.

The chimeric protein comprising an adjunct moiety, e.g., a VWF fragment,and a FVIII protein can be used prophylactically to treat a subject witha hemostatic disorder. The chimeric protein of the invention can be usedto treat an acute bleeding episode in a subject with a hemostaticdisorder. In another embodiment, the hemostatic disorder can be theresult of a defective clotting factor, e.g., von Willebrand's factor. Inone embodiment, the hemostatic disorder is an inherited disorder. Inanother embodiment, the hemostatic disorder is an acquired disorder. Theacquired disorder can result from an underlying secondary disease orcondition. The unrelated condition can be, as an example, but not as alimitation, cancer, an auto-immune disease, or pregnancy. The acquireddisorder can result from old age or from medication to treat anunderlying secondary disorder (e.g. cancer chemotherapy).

The invention also relates to methods of treating a subject that doesnot have a congenital hemostatic disorder, but has a secondary diseaseor condition resulting in acquisition of a hemostatic disorder, e.g.,due to development of an anti-FVIII antibody or a surgery. The inventionthus relates to a method of treating a subject in need of a generalhemostatic agent comprising administering a therapeutically effectiveamount of the chimeric protein comprising the VWF fragment and a FVIIIprotein prepared by the present methods.

The present invention is also related to methods of reducingimmunogenicity of FVIII or inducing less immunogenicity against FVIIIcomprising administering an effective amount of the VWF fragment, thechimeric proteins described herein, or the polynucleotides encoding thesame.

In one embodiment, the subject in need of a general hemostatic agent isundergoing, or is about to undergo, surgery. The chimeric proteincomprising the VWF fragment and a FVIII protein can be administeredprior to, during, or after surgery as a prophylactic regimen. Thechimeric protein comprising the VWF fragment and a FVIII protein can beadministered prior to, during, or after surgery to control an acutebleeding episode.

The chimeric protein comprising the VWF fragment and a FVIII protein canbe used to treat a subject having an acute bleeding episode who does nothave a hemostatic disorder. The acute bleeding episode can result fromsevere trauma, e.g., surgery, an automobile accident, wound, lacerationgun shot, or any other traumatic event resulting in uncontrolledbleeding. Non limiting examples of bleeding episodes include a bleedingcoagulation disorder, hemarthrosis, muscle bleed, oral bleed,hemorrhage, hemorrhage into muscles, oral hemorrhage, trauma, traumacapitis, gastrointestinal bleeding, intracranial hemorrhage,intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture,central nervous system bleeding, bleeding in the retropharyngeal space,bleeding in the retroperitoneal space, bleeding in the illiopsoassheath, and any combinations thereof.

In prophylactic applications, one or more compositions containing thechimeric protein or the VWF fragment of the invention or a cocktailthereof are administered to a patient not already in the disease stateto enhance the patient's resistance or reduce symptoms associated with adisease or disorder. Such an amount is defined to be a “prophylacticeffective dose.” In therapeutic applications, a relatively high dosage(e.g., from about 1 to 400 mg/kg of polypeptide per dose, with dosagesof from 5 to 25 mg being more commonly used for radioimmunoconjugatesand higher doses for cytotoxin-drug modified polypeptides) at relativelyshort intervals is sometimes required until progression of the diseaseis reduced or terminated, and until the patient shows partial orcomplete amelioration of symptoms of disease. Thereafter, the patientcan be administered a prophylactic regime.

In some embodiments, a chimeric protein, a VWF fragment, or acomposition of the invention is used for on-demand treatment, whichincludes treatment for a bleeding episode, hemarthrosis, muscle bleed,oral bleed, hemorrhage, hemorrhage into muscles, oral hemorrhage,trauma, trauma capitis (head trauma), gastrointestinal bleeding,intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracichemorrhage, bone fracture, central nervous system bleeding, bleeding inthe retropharyngeal space, bleeding in the retroperitoneal space, orbleeding in the illiopsoas sheath. The subject may be in need ofsurgical prophylaxis, peri-operative management, or treatment forsurgery. Such surgeries include, e.g., minor surgery, major surgery,tooth extraction, tonsillectomy, inguinal herniotomy, synovectomy, totalknee replacement, craniotomy, osteosynthesis, trauma surgery,intracranial surgery, intra-abdominal surgery, intrathoracic surgery, orjoint replacement surgery.

In one embodiment, the chimeric protein comprising the VWF fragment anda FVIII protein is administered intravenously, subcutaneously,intramuscularly, or via any mucosal surface, e.g., orally, sublingually,buccally, nasally, rectally, vaginally or via pulmonary route. Thechimeric protein comprising the VWF fragment and a FVIII protein can beimplanted within or linked to a biopolymer solid support that allows forthe slow release of the chimeric protein to the site of bleeding orimplanted into bandage/dressing. The dose of the chimeric proteincomprising the VWF fragment and a FVIII protein will vary depending onthe subject and upon the particular route of administration used.Dosages can range from 0.1 to 100,000 μg/kg body weight. In oneembodiment, the dosing range is 0.1-1,000 μg/kg. In another embodiment,the dosing range is 0.1-500 μg/kg. The protein can be administeredcontinuously or at specific timed intervals. In vitro assays may beemployed to determine optimal dose ranges and/or schedules foradministration. In vitro assays that measure clotting factor activityare known in the art, e.g., STA-CLOT VIIa-rTF clotting assay or ROTEMclotting assay. Additionally, effective doses may be extrapolated fromdose-response curves obtained from animal models, e.g., a hemophiliacdog (Mount et al. 2002, Blood 99(8):2670).

Having now described the present invention in detail, the same will bemore clearly understood by reference to the following examples, whichare included herewith for purposes of illustration only and are notintended to be limiting of the invention. All patents and publicationsreferred to herein are expressly incorporated by reference.

EXAMPLES

Throughout the examples, the following materials and methods were usedunless otherwise stated.

Materials and Methods

In general, the practice of the present invention employs, unlessotherwise indicated, conventional techniques of chemistry, biophysics,molecular biology, recombinant DNA technology, immunology (especially,e.g., antibody technology), and standard techniques in electrophoresis.See, e.g., Sambrook, Fritsch and Maniatis, Molecular Cloning: ColdSpring Harbor Laboratory Press (1989); Antibody Engineering Protocols(Methods in Molecular Biology), 510, Paul, S., Humana Pr (1996);Antibody Engineering: A Practical Approach (Practical Approach Series,169), McCafferty, Ed., Irl Pr (1996); Antibodies: A Laboratory Manual,Harlow et al., CS.H.L. Press, Pub. (1999); and Current Protocols inMolecular Biology, eds. Ausubel et al., John Wiley & Sons (1992).

Example 1: Cloning Different VWF Domains (FIG. 1)

(a) Cloning pSYN-VWF-001, 002, 003 and 004

pSYN-VWF-001 through 004 contain nucleotide sequences encoding VWFfragments, which are amino acids 1-276 (001), amino acids 1-477 (002),amino acids 1-511 (003) and amino acids 1-716 (004)VWF-D′D3A proteinsequence. Amino acid numbering represents the mature VWF sequencewithout propeptide and corresponds to amino acids 764-1039 (001), aminoacids 764-1240 (002), amino acids 764-1274 (003), and amino acids764-1479 (004) of SEQ ID NO: 2, respectively. All four constructs havethe FVIII signal peptide at N-terminus, which allows proper secretion ofthe synthesized protein and followed by a 6×His tag at C-terminus, whichis used for protein purification. Above constructs were synthesized byusing following primer combinations:

pSYN VWF-001: ESC48-Fwd-VWF-D′D3 with VIII (SEQ ID NO: 57)signal and BsiW1 site TCGCGACGTACGGCCGCCACCATGCAAATAGAGCTCTCCACCTGCTTCTTTCTGTGCC TTTTGCGATTCTGCTTTAGCCTATCCTGT CGGCCCCCCATGESC50-Rev-VWF-partial D′D3 (1-276 amino acid) with 6 His and Not1 site(SEQ ID NO: 58) TGACCTCGAGCGGCCGCTCAGTGGTGATGGTGATGATGCAGAGGCACTTTTCTGGTG TCAGCACACTG pSYN VWF-002:ESC48-Fwd-VWF-D′D3 with VIII signal and BsiW1 site (SEQ ID NO: 59)TCGCGACGTACGGCCGCCACCATGCAAAT AGAGCTCTCCACCTGCTTCTTTCTGTGCCTTTTGCGATTCTGCTTTAGCCTATCCTGT CGGCCCCCCATG ESC51-Rev-VWF D′D3 (1-477amino acid) with 6His and Not1 site (SEQ ID NO: 60)TGACCTCGAGCGGCCGCTCAGTGGTGATG GTGATGATGCGGCTCCTGGCAGGCTTCACAGGTGAGGTTGACAAC pSYN VWF-003: ESC48-Fwd-VWF-D′D3 with VIIIsignal and BsiW1 site (SEQ ID NO: 61) TCGCGACGTACGGCCGCCACCATGCAAATAGAGCTCTCCACCTGCTTCTTTCTGTGCC TTTTGCGATTCTGCTTTAGCCTATCCTGT CGGCCCCCCATGESC52-Rev-VWF-D′D3 Partial A1 (1-511 amino acids) with 6Hisand Not1 site (SEQ ID NO: 62) TGACCTCGAGCGGCCGCTCAGTGGTGATGGTGATGATGCCTGCTGCAGTAGAAATCG TGCAACGGCGGTTC pSYN VWF-004:ESC48-Fwd-VWF-D′D3 with VIII signal and BsiW1 site (SEQ ID NO: 63)TCGCGACGTACGGCCGCCACCATGCAAAT AGAGCTCTCCACCTGCTTCTTTCTGTGCCTTTTGCGATTCTGCTTTAGCCTATCCTGT CGGCCCCCCATG ESC53-Rev-VWF-D′D3A1 (1-716amino acids) with 6His and Not1 site (SEQ ID NO: 64)TGACCTCGAGCGGCCGCTCAGTGGTGATG GTGATGATGGCCCACAGTGACTTGTGCC ATGTGGGGProteins from VWF-001, 002, 003 and 004 constructs are supposed toexists as a monomer.

A 50 μl PCR reaction was carried out with ESC 48/ESC50, ESC 48/ESC 51,ESC 48/ESC52, ESC48/ESC53 primer combinations and full length VWFplasmid as the template, using the 2 step PCR amplification cycle: 94°C. 2 minutes; 21 cycles of (96° C. 30 seconds, 68° C. 2 minute). Correctsized bands (-960 bp for VWF 001; 1460 for VWF 002, 1520 bp for VWF 003;and 2150 bp for VWF 004) were gel purified with a Gel Extraction kit(Qiagen, Valencia, Calif.) and cloned into the BsiWI and Not1restriction sites of pcDNA 4 to generate pSYN-VWF 001,002,003 and 004,respectively.

(b) Cloning pSYN-VWF-006

pSYN-VWF-006 contains D1D2D′D3-CK (cysteine knot) domain of VWF. Toclone this construct, synthesis of DNA fragment containing a portion ofD3 domain and CK domain was outsourced (Genscript-sequence id number122026, shown below). A fragment of Genscript construct was sub-clonedinto the BamH1/EcoRV digested pSYN-VWF 008, i.e., the vector codingfull-length VWF.

Genscript-Sequence number-122026 (SEQ ID NO: 65)GGATCCTAGTGGGGAATAAGGGATGCAGCCACCCCTCAGTGAAATGCAAGAAACGGGTCACCATCCTGGTGGAGGGAGGAGAGATTGAGCTGTTTGACGGGGAGGTGAATGTGAAGAGGCCCATGAAGGATGAGACTCACTTTGAGGTGGTGGAGTCTGGCCGGTACATCATTCTGCTGCTGGGCAAAGCCCTCTCCGTGGTCTGGGACCGCCACCTGAGCATCTCCGTGGTCCTGAAGCAGACATACCAGGAGAAAGTGTGTGGCCTGTGTGGGAATTTTGATGGCATCCAGAACAATGACCTCACCAGCAGCAACCTCCAAGTGGAGGAAGACCCTGTGGACTTTGGGAACTCCTGGAAAGTGAGCTCGCAGTGTGCTGACACCAGAAAAGTGCCTCTGGACTCATCCCCTGCCACCTGCCATAACAACATCATGAAGCAGACGATGGTGGATTCCTCCTGTAGAATCCTTACCAGTGACGTCTTCCAGGACTGCAACAAGCTGGTGGACCCCGAGCCATATCTGGATGTCTGCATTTACGACACCTGCTCCTGTGAGTCCATTGGGGACTGCGCCTGCTTCTGCGACACCATTGCTGCCTATGCCCACGTGTGTGCCCAGCATGGCAAGGTGGTGACCTGGAGGACGGCCACATTGTGCCCCCAGAGCTGCGAGGAGAGGAATCTCCGGGAGAACGGGTATGAGTGTGAGTGGCGCTATAACAGCTGTGCACCTGCCTGTCAAGTCACGTGTCAGCACCCTGAGCCACTGGCCTGCCCTGTGCAGTGTGTGGAGGGCTGCCATGCCCACTGCCCTCCAGGGAAAATCCTGGATGAGCTTTTGCAGACCTGCGTTGACCCTGAAGACTGTCCAGTGTGTGAGGTGGCTGGCCGGCGTTTTGCCTCAGGAAAGAAAGTCACCTTGAATCCCAGTGACCCTGAGCACTGCCAGATTTGCCACTGTGATGTTGTCAACCTCACCTGTGAAGCCTGCCAGGAGCCGGGAGGCCTGGTGGTGCCTCCCACAGATGCCCCGGTGAGCCCCACCACTCTGTATGTGGATGAGACGCTCCAGGATGGCTGTGATACTCACTTCTGCAAGGTCAATGAGAGAGGAGAGTACTTCTGGGAGAAGAGGGTCACAGGCTGCCCACCCTTTGATGAACACAAGTGTCTTGCTGAGGGAGGTAAAATTATGAAAATTCCAGGCACCTGCTGTGACACATGTGAGGAGCCTGAGTGCAACGACATCACTGCCAGGCTGCAGTATGTCAAGGTGGGAAGCTGTAAGTCTGAAGTAGAGGTGGATATC

(c) Cloning pSYN-VWF-009, 010, 011, 012 and 013

pSYN VWF 008 construct contains the full-length VWF sequence in pcDNA3.1 (amino acids 1-2813 of SEQ ID NO: 2). It includes 763 amino acidpropeptide (i.e., D1D2 domains) followed by remaining 2050 amino acidssequence of mature VWF. pSYN-VWF-009, 010, 011 and 012 contain the samecoding sequences as VWF 001, 002, 003 and 004, respectively, butadditionally has D1D2 domains (VWF propeptide) at the N-terminus insteadof the FVIII signal peptide. pSYN-VWF-008 has a BamH1 site at Arg907 andNot1 site at the end of coding region (after stop codon). pSYN-VWF-008,001, 002, 003 and 004 were digested with BamH1 and Not1 restrictionenzymes. Inserts from pSYN-VWF-001 (423 bp), pSYN-VWF-002 (1026 bp),pSYN-VWF-003 (1128 bp) and pSYN-VWF-004 (1743 bp) were ligated intobamH1/Not1 digested pSYN-VWF-008 (8242 bp) to obtain pSYN-VWF-009(D1D2D′D3: amino acid 1-1039 of SEQ ID NO: 2); pSYN-VWF-010 (D1D2D′D3:amino acid 1-1240 of SEQ ID NO: 2); pSYN-VWF-011 (D1D2D′D3: amino acid1-1274 of SEQ ID NO: 2); pSYN-VWF-012 (D1D2D′D3: amino acid 1-1479). All4 constructs have 6×His tag at the C-terminus. In transfected cells,pSYN-VWF-009, 010, 011, and 012 are synthesized with propeptide, but dueto intracellular processing, the secreted products do not contain anypropeptide (D1D2). The protein expressed from the VWF-009 constructexists as a monomer and the proteins expressed from the VWF-010, 011,and 012 constructs are supposed to exist as dimers, as shown in FIG. 6and FIG. 7 using VWF-009 and VWF-010 as examples, respectively.

pSYN-VWF-010 was used to generate pSYN-VWF-013, which has two pointmutations at C336A and C379A corresponding to SEQ ID NO: 73 (amino acidnumbering represents mature VWF sequence without D1D2 domain-VWFsequence 2). These mutations are predicted to prevent dimerization ofVWF D′D3 domain.

(d) Cloning pSYN-VWF-025 and 029

pSYN-VWF-025 contains wild type D1D2D′D3 sequences of full-length VWF inpLIVE vector while pSYN-VWF-029 contains D1D2D′D3 domains withC336A/C379A mutations in pLIVE vector. For cloning pSYN-VWF-025 and 029,the following primer combination was used:

ESC 89-fwd with NheI site = (SEQ ID NO: 66)CTCACTATAGGGAGACCCAAGCTGGCTAGCCG  ESC 91-rev with SalI= (SEQ ID NO: 67)CTGGATCCCGGGAGTCGACTCGTCAGTGGTGATGGTGATGATG

A 50 μl PCR reaction was carried out with ESC 89/ESC91 primercombinations and either pSYN-VWF-010 (for pSYN-VWF-025) or pSYN-VWF-013(for pSYN-VWF-029) plasmid as the template using the 3 step PCRamplification cycle: 94° C.—2 minutes; 21 cycles of (96° C.—30 seconds,55° C.—30 second, 68° C.—4 minutes). The expected sized band (˜3800 bp)was gel purified with a Gel Extraction kit (Qiagen, Valencia, Calif.)and cloned into the Nhe1 and Sal1 restriction sites of pLIVE-Mirusvector (Invitrogen, Carlsbad, Calif.) to generate pSYN-VWF-025 and 029.

(e) Cloning pSYN-VWF-031

pSYN-VWF-031 is a D1D2D′D3 (C336A/C379A)-Fc construct which has a 48amino acid long thrombin cleavable linker (8×GGGGS (SEQ ID NO:110)+thrombin site) in between the VWF D1D2D′D3 (C336A/C379A) and the Fcsequences. To make this construct, VWF-Fc region was amplified fromconstruct pSYN-FVIII-064 (refer FVIII-VWF construct below).pSYN-FVIII-VWF was digested with Xba1 and Nhe1. The resulting insertregion of 4165 bp, containing the VWF fragment and Fc region, was usedas a template for amplifying the VWF and Fc region by primercombinations LW 22/LW23.

LW 22-FWD-VWF-DD3 with FVIII signal sequence and BsiWI site(SEQ ID NO: 68)GCGCCGGCCGTACGATGCAAATAGAGCTCTCCACCTGCTTCTTTCTGTGCCTTTTGCGATTCTGCTTTAGCCTATCCTGTCGGCCCCCCATGLW 23-Rev-Fc with stop codon and NotI site (SEQ ID NO: 69)TCATCAATGTATCTTATCATGTCTGAATTCGCGGCCGCTCATTTACC Nucleotide sequence of VWF 031 (SEQ ID NO: 108) 1ATGATTCCTG CCAGATTTGC CGGGGTGCTG CTTGCTCTGG CCCTCATTTT 51GCCAGGGACC CTTTGTGCAG AAGGAACTCG CGGCAGGTCA TCCACGGCCC 101GATGCAGCCT TTTCGGAAGT GACTTCGTCA ACACCTTTGA TGGGAGCATG 151TACAGCTTTG CGGGATACTG CAGTTACCTC CTGGCAGGGG GCTGCCAGAA 201ACGCTCCTTC TCGATTATTG GGGACTTCCA GAATGGCAAG AGAGTGAGCC 251TCTCCGTGTA TCTTGGGGAA TTTTTTGACA TCCATTTGTT TGTCAATGGT 301ACCGTGACAC AGGGGGACCA AAGAGTCTCC ATGCCCTATG CCTCCAAAGG 351GCTGTATCTA GAAACTGAGG CTGGGTACTA CAAGCTGTCC GGTGAGGCCT 401ATGGCTTTGT GGCCAGGATC GATGGCAGCG GCAACTTTCA AGTCCTGCTG 451TCAGACAGAT ACTTCAACAA GACCTGCGGG CTGTGTGGCA ACTTTAACAT 501CTTTGCTGAA GATGACTTTA TGACCCAAGA AGGGACCTTG ACCTCGGACC 551CTTATGACTT TGCCAACTCA TGGGCTCTGA GCAGTGGAGA ACAGTGGTGT 601GAACGGGCAT CTCCTCCCAG CAGCTCATGC AACATCTCCT CTGGGGAAAT 651GCAGAAGGGC CTGTGGGAGC AGTGCCAGCT TCTGAAGAGC ACCTCGGTGT 701TTGCCCGCTG CCACCCTCTG GTGGACCCCG AGCCTTTTGT GGCCCTGTGT 751GAGAAGACTT TGTGTGAGTG TGCTGGGGGG CTGGAGTGCG CCTGCCCTGC 801CCTCCTGGAG TACGCCCGGA CCTGTGCCCA GGAGGGAATG GTGCTGTACG 851GCTGGACCGA CCACAGCGCG TGCAGCCCAG TGTGCCCTGC TGGTATGGAG 901TATAGGCAGT GTGTGTCCCC TTGCGCCAGG ACCTGCCAGA GCCTGCACAT 951CAATGAAATG TGTCAGGAGC GATGCGTGGA TGGCTGCAGC TGCCCTGAGG 1001GACAGCTCCT GGATGAAGGC CTCTGCGTGG AGAGCACCGA GTGTCCCTGC 1051GTGCATTCCG GAAAGCGCTA CCCTCCCGGC ACCTCCCTCT CTCGAGACTG 1101CAACACCTGC ATTTGCCGAA ACAGCCAGTG GATCTGCAGC AATGAAGAAT 1151GTCCAGGGGA GTGCCTTGTC ACTGGTCAAT CCCACTTCAA GAGCTTTGAC 1201AACAGATACT TCACCTTCAG TGGGATCTGC CAGTACCTGC TGGCCCGGGA 1251TTGCCAGGAC CACTCCTTCT CCATTGTCAT TGAGACTGTC CAGTGTGCTG 1301ATGACCGCGA CGCTGTGTGC ACCCGCTCCG TCACCGTCCG GCTGCCTGGC 1351CTGCACAACA GCCTTGTGAA ACTGAAGCAT GGGGCAGGAG TTGCCATGGA 1401TGGCCAGGAC ATCCAGCTCC CCCTCCTGAA AGGTGACCTC CGCATCCAGC 1451ATACAGTGAC GGCCTCCGTG CGCCTCAGCT ACGGGGAGGA CCTGCAGATG 1501GACTGGGATG GCCGCGGGAG GCTGCTGGTG AAGCTGTCCC CCGTCTATGC 1551CGGGAAGACC TGCGGCCTGT GTGGGAATTA CAATGGCAAC CAGGGCGACG 1601ACTTCCTTAC CCCCTCTGGG CTGGCGGAGC CCCGGGTGGA GGACTTCGGG 1651AACGCCTGGA AGCTGCACGG GGACTGCCAG GACCTGCAGA AGCAGCACAG 1701CGATCCCTGC GCCCTCAACC CGCGCATGAC CAGGTTCTCC GAGGAGGCGT 1751GCGCGGTCCT GACGTCCCCC ACATTCGAGG CCTGCCATCG TGCCGTCAGC 1801CCGCTGCCCT ACCTGCGGAA CTGCCGCTAC GACGTGTGCT CCTGCTCGGA 1851CGGCCGCGAG TGCCTGTGCG GCGCCCTGGC CAGCTATGCC GCGGCCTGCG 1901CGGGGAGAGG CGTGCGCGTC GCGTGGCGCG AGCCAGGCCG CTGTGAGCTG 1951AACTGCCCGA AAGGCCAGGT GTACCTGCAG TGCGGGACCC CCTGCAACCT 2001GACCTGCCGC TCTCTCTCTT ACCCGGATGA GGAATGCAAT GAGGCCTGCC 2051TGGAGGGCTG CTTCTGCCCC CCAGGGCTCT ACATGGATGA GAGGGGGGAC 2101TGCGTGCCCA AGGCCCAGTG CCCCTGTTAC TATGACGGTG AGATCTTCCA 2151GCCAGAAGAC ATCTTCTCAG ACCATCACAC CATGTGCTAC TGTGAGGATG 2201GCTTCATGCA CTGTACCATG AGTGGAGTCC CCGGAAGCTT GCTGCCTGAC 2251GCTGTCCTCA GCAGTCCCCT GTCTCATCGC AGCAAAAGGA GCCTATCCTG 2301TCGGCCCCCC ATGGTCAAGC TGGTGTGTCC CGCTGACAAC CTGCGGGCTG 2351AAGGGCTCGA GTGTACCAAA ACGTGCCAGA ACTATGACCT GGAGTGCATG 2401AGCATGGGCT GTGTCTCTGG CTGCCTCTGC CCCCCGGGCA TGGTCCGGCA 2451TGAGAACAGA TGTGTGGCCC TGGAAAGGTG TCCCTGCTTC CATCAGGGCA 2501AGGAGTATGC CCCTGGAGAA ACAGTGAAGA TTGGCTGCAA CACTTGTGTC 2551TGTCGGGACC GGAAGTGGAA CTGCACAGAC CATGTGTGTG ATGCCACGTG 2601CTCCACGATC GGCATGGCCC ACTACCTCAC CTTCGACGGG CTCAAATACC 2651TGTTCCCCGG GGAGTGCCAG TACGTTCTGG TGCAGGATTA CTGCGGCAGT 2701AACCCTGGGA CCTTTCGGAT CCTAGTGGGG AATAAGGGAT GCAGCCACCC 2751CTCAGTGAAA TGCAAGAAAC GGGTCACCAT CCTGGTGGAG GGAGGAGAGA 2801TTGAGCTGTT TGACGGGGAG GTGAATGTGA AGAGGCCCAT GAAGGATGAG 2851ACTCACTTTG AGGTGGTGGA GTCTGGCCGG TACATCATTC TGCTGCTGGG 2901CAAAGCCCTC TCCGTGGTCT GGGACCGCCA CCTGAGCATC TCCGTGGTCC 2951TGAAGCAGAC ATACCAGGAG AAAGTGTGTG GCCTGTGTGG GAATTTTGAT 3001GGCATCCAGA ACAATGACCT CACCAGCAGC AACCTCCAAG TGGAGGAAGA 3051CCCTGTGGAC TTTGGGAACT CCTGGAAAGT GAGCTCGCAG TGTGCTGACA 3101CCAGAAAAGT GCCTCTGGAC TCATCCCCTG CCACCTGCCA TAACAACATC 3151ATGAAGCAGA CGATGGTGGA TTCCTCCTGT AGAATCCTTA CCAGTGACGT 3201CTTCCAGGAC TGCAACAAGC TGGTGGACCC CGAGCCATAT CTGGATGTCT 3251GCATTTACGA CACCTGCTCC TGTGAGTCCA TTGGGGACTG CGCCGCATTC 3301TGCGACACCA TTGCTGCCTA TGCCCACGTG TGTGCCCAGC ATGGCAAGGT 3351GGTGACCTGG AGGACGGCCA CATTGTGCCC CCAGAGCTGC GAGGAGAGGA 3401ATCTCCGGGA GAACGGGTAT GAGGCTGAGT GGCGCTATAA CAGCTGTGCA 3451CCTGCCTGTC AAGTCACGTG TCAGCACCCT GAGCCACTGG CCTGCCCTGT 3501GCAGTGTGTG GAGGGCTGCC ATGCCCACTG CCCTCCAGGG AAAATCCTGG 3551ATGAGCTTTT GCAGACCTGC GTTGACCCTG AAGACTGTCC AGTGTGTGAG 3601GTGGCTGGCC GGCGTTTTGC CTCAGGAAAG AAAGTCACCT TGAATCCCAG 3651TGACCCTGAG CACTGCCAGA TTTGCCACTG TGATGTTGTC AACCTCACCT 3701GTGAAGCCTG CCAGGAGCCG ATATCTGGCG GTGGAGGTTC CGGTGGCGGG 3751GGATCCGGCG GTGGAGGTTC CGGCGGTGGA GGTTCCGGTG GCGGGGGATC 3801CGGTGGCGGG GGATCCCTGG TCCCCCGGGG CAGCGGCGGT GGAGGTTCCG 3851GTGGCGGGGG ATCCGACAAA ACTCACACAT GCCCACCGTG CCCAGCTCCA 3901GAACTCCTGG GCGGACCGTC AGTCTTCCTC TTCCCCCCAA AACCCAAGGA 3951CACCCTCATG ATCTCCCGGA CCCCTGAGGT CACATGCGTG GTGGTGGACG 4001TGAGCCACGA AGACCCTGAG GTCAAGTTCA ACTGGTACGT GGACGGCGTG 4051GAGGTGCATA ATGCCAAGAC AAAGCCGCGG GAGGAGCAGT ACAACAGCAC 4101GTACCGTGTG GTCAGCGTCC TCACCGTCCT GCACCAGGAC TGGCTGAATG 4151GCAAGGAGTA CAAGTGCAAG GTCTCCAACA AAGCCCTCCC AGCCCCCATC 4201GAGAAAACCA TCTCCAAAGC CAAAGGGCAG CCCCGAGAAC CACAGGTGTA 4251CACCCTGCCC CCATCCCGGG ATGAGCTGAC CAAGAACCAG GTCAGCCTGA 4301CCTGCCTGGT CAAAGGCTTC TATCCCAGCG ACATCGCCGT GGAGTGGGAG 4351AGCAATGGGC AGCCGGAGAA CAACTACAAG ACCACGCCTC CCGTGTTGGA 4401CTCCGACGGC TCCTTCTTCC TCTACAGCAA GCTCACCGTG GACAAGAGCA 4451GGTGGCAGCA GGGGAACGTC TTCTCATGCT CCGTGATGCA TGAGGCTCTG 4501CACAACCACT ACACGCAGAA GAGCCTCTCC CTGTCTCCGG GTAAATGAProtein sequence of VWF 031 (SEQ ID NO: 109) 1MIPARFAGVL LALALILPGT LCAEGTRGRS STARCSLFGS DFVNTFDGSM 51YSFAGYCSYL LAGGCQKRSF SIIGDFQNGK RVSLSVYLGE FFDIHLFVNG 101TVTQGDQRVS MPYASKGLYL ETEAGYYKLS GEAYGFVARI DGSGNFQVLL 151SDRYFNKTCG LCGNFNIFAE DDFMTQEGTL TSDPYDFANS WALSSGEQWC 201ERASPPSSSC NISSGEMQKG LWEQCQLLKS TSVFARCHPL VDPEPFVALC 251EKTLCECAGG LECACPALLE YARTCAQEGM VLYGWTDHSA CSPVCPAGME 301YRQCVSPCAR TCQSLHINEM CQERCVDGCS CPEGQLLDEG LCVESTECPC 351VHSGKRYPPG TSLSRDCNTC ICRNSQWICS NEECPGECLV TGQSHFKSFD 401NRYFTFSGIC QYLLARDCQD HSFSIVIETV QCADDRDAVC TRSVTVRLPG 451LHNSLVKLKH GAGVAMDGQD IQLPLLKGDL RIQHTVTASV RLSYGEDLQM 501DWDGRGRLLV KLSPVYAGKT CGLCGNYNGN QGDDFLTPSG LAEPRVEDFG 551NAWKLHGDCQ DLQKQHSDPC ALNPRMTRFS EEACAVLTSP TFEACHRAVS 601PLPYLRNCRY DVCSCSDGRE CLCGALASYA AACAGRGVRV AWREPGRCEL 651NCPKGQVYLQ CGTPCNLTCR SLSYPDEECN EACLEGCFCP PGLYMDERGD 701CVPKAQCPCY YDGEIFQPED IFSDHHTMCY CEDGFMHCTM SGVPGSLLPD 751AVLSSPLSHR SKRSLSCRPP MVKLVCPADN LRAEGLECTK TCQNYDLECM 801SMGCVSGCLC PPGMVRHENR CVALERCPCF HQGKEYAPGE TVKIGCNTCV 851CRDRKWNCTD HVCDATCSTI GMAHYLTFDG LKYLFPGECQ YVLVQDYCGS 901NPGTFRILVG NKGCSHPSVK CKKRVTILVE GGEIELFDGE VNVKRPMKDE 951THFEVVESGR YIILLLGKAL SVVWDRHLSI SVVLKQTYQE KVCGLCGNFD 1001GIQNNDLTSS NLQVEEDPVD FGNSWKVSSQ CADTRKVPLD SSPATCHNNI 1051MKQTMVDSSC RILTSDVFQD CNKLVDPEPY LDVCIYDTCS CESIGDCAAF 1101CDTIAAYAHV CAQHGKVVTW RTATLCPQSC EERNLRENGY EAEWRYNSCA 1151PACQVTCQHP EPLACPVQCV EGCHAHCPPG KILDELLQTC VDPEDCPVCE 1201VAGRRFASGK KVTLNPSDPE HCQICHCDVV NLTCEACQEP ISGGGGSGGG 1251GSGGGGSGGG GSGGGGSGGG GSLVPRGSGG GGSGGGGSDK THTCPPCPAP 1301ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE VKFNWYVDGV 1351EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKALPAPI 1401EKTISKAKGQ PREPQVYTLP PSRDELTKNQ VSLTCLVKGF YPSDIAVEWE 1451SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV FSCSVMHEAL 1501HNHYTQKSLS LSPGK*

DNA construct Linker between VWF and Fc VWF03573 aa= IS{11X(GGGGS)} LVPRGSGGGGSGGGGS (SEQ ID NO: 96) VWF03698 aa= IS{16X(GGGGS)} LVPRGSGGGGSGGGGS (SEQ ID NO: 97)VWF = D’D3 (l-477 aa with C336A/C379A)

The PCR product obtained from LW22/LW23 amplification (-2300 bp) wascloned in BsiW1/Not1 digested pSYN-VWF-002 to obtain pSYN-VWF-014intermediate. pSYN-VWF-014 contains FVIII signal peptide-D′D3-20 aminoacid thrombin cleavable linker followed by the Fc region.

To generate the D1D2D′D3-Fc construct, the D1D2D′D3 region was amplifiedfrom pSYN-VWF-013 using primer combination LW24/LW27 by standard PCRmethod.

LW24-Fwd-VWF D1D2D′D3 cloning oligo with BsiW1 site (SEQ ID NO: 70)GCGCCGGCCGTACGATGATTCCTGCC AGATTTGCCGGGGTGLW27-Rev-VWF D′D3 oligo with EcoRV (SEQ ID NO: 71)CCACCGCCAGATATCGGCTCCTGGCAGGCTTCACAGGTGAG

The PCR product obtained from LW22/LW23 amplification (-3750 bp) wascloned in BsiW1/EcoRV digested pSYN-VWF-014 to obtain pSYN-VWF-015intermediate. The linker length between the VWF fragment and Fc regionwas changed to obtain pSYN-VWF-031.

Full length VWF protein sequence is shown at Table 1.

VWF-D1D2D′D3 protein sequence 1b (SEQ ID NO: 72) 1MIPARFAGVL LALALILPGT LCAEGTRGRS STARCSLFGS DFVNTFDGSM 51YSFAGYCSYL LAGGCQKRSF SIIGDFQNGK RVSLSVYLGE FFDIHLFVNG 101TVTQGDQRVS MPYASKGLYL ETEAGYYKLS GEAYGFVARI DGSGNFQVLL 151SDRYFNKTCG LCGNFNIFAE DDFMTQEGTL TSDPYDFANS WALSSGEQWC 201ERASPPSSSC NISSGEMQKG LWEQCQLLKS TSVFARCHPL VDPEPFVALC 251EKTLCECAGG LECACPALLE YARTCAQEGM VLYGWTDHSA CSPVCPAGME 301YRQCVSPCAR TCQSLHINEM CQERCVDGCS CPEGQLLDEG LCVESTECPC 351VHSGKRYPPG TSLSRDCNTC ICRNSQWICS NEECPGECLV TGQSHFKSFD 401NRYFTFSGIC QYLLARDCQD HSFSIVIETV QCADDRDAVC TRSVTVRLPG 451LHNSLVKLKH GAGVAMDGQD IQLPLLKGDL RIQHTVTASV RLSYGEDLQM 501DWDGRGRLLV KLSPVYAGKT CGLCGNYNGN QGDDFLTPSG LAEPRVEDFG 551NAWKLHGDCQ DLQKQHSDPC ALNPRMTRFS EEACAVLTSP TFEACHRAVS 601PLPYLRNCRY DVCSCSDGRE CLCGALASYA AACAGRGVRV AWREPGRCEL 651NCPKGQVYLQ CGTPCNLTCR SLSYPDEECN EACLEGCFCP PGLYMDERGD 701CVPKAQCPCY YDGEIFQPED IFSDHHTMCY CEDGFMHCTM SGVPGSLLPD 751AVLSSPLSHR SKRSLSCRPP MVKLVCPADN LRAEGLECTK TCQNYDLECM 801SMGCVSGCLC PPGMVRHENR CVALERCPCF HQGKEYAPGE TVKIGCNTCV 851CRDRKWNCTD HVCDATCSTI GMAHYLTFDG LKYLFPGECQ YVLVQDYCGS 901NPGTFRILVG NKGCSHPSVK CKKRVTILVE GGEIELFDGE VNVKRPMKDE 951THFEVVESGR YIILLLGKAL SVVWDRHLSI SVVLKQTYQE KVCGLCGNFD 1001GIQNNDLTSS NLQVEEDPVD FGNSWKVSSQ CADTRKVPLD SSPATCHNNI 1051MKQTMVDSSC RILTSDVFQD CNKLVDPEPY LDVCIYDTCS CESIGDCACF 1101CDTIAAYAHV CAQHGKVVTW RTATLCPQSC EERNLRENGY ECEWRYNSCA 1151PACQVTCQHP EPLACPVQCV EGCHAHCPPG KILDELLQTC VDPEDCPVCE 1201VAGRRFASGK KVTLNPSDPE HCQICHCDW NLTCEACQEP* VWF-D′D3 protein sequence 2(SEQ ID NO: 73) 1 SLSCRPPMVK LVCPADNLRA EGLECTKTCQ NYDLECMSMG CVSGCLCPPG51 MVRHENRCVA LERCPCFHQG KEYAPGETVK IGCNTCVCRD RKWNCTDHVC 101DATCSTIGMA HYLTFDGLKY LFPGECQYVL VQDYCGSNPG TFRILVGNKG 151CSHPSVKCKK RVTILVEGGE IELFDGEVNV KRPMKDETHF EVVESGRYII 201LLLGKALSVV WDRHLSISVV LKQTYQEKVC GLCGNFDGIQ NNDLTSSNLQ 251VEEDPVDFGN SWKVSSQCAD TRKVPLDSSP ATCHNNIMKQ TMVDSSCRIL 301TSDVFQDCNK LVDPEPYLDV CIYDTCSCES IGDCACFCDT IAAYAHVCAQ 351HGKVVTWRTA TLCPQSCEER NLRENGYECE WRYNSCAPAC QVTCQHPEPL 401ACPVQCVEGC HAHCPPGKIL DELLQTCVDP EDCPVCEVAG RRFASGKKVT 451LNPSDPEHCQ ICHCDWNLT CEACQEP

Example 2: Heterodimeric Constructs Comprising FVIII-Fc and VWF-D′D3Domain at the Amino Terminus of the Second Fc Chain (FVIII-VWF-FcHeterodimer, FIG. 2)

(a) Cloning of pSYN-FVIII-064

The FVIII-064 plasmid comprises a single chain FC (scFc) scaffold withenzyme cleavage sites which are processed during synthesis in a cell.The construct has a FVIII binding domain of full-length VWF (D′D3).

Plasmid (pSYN-FVIII-064) was designed for the expression FVIII-Fc andVWF-Fc heterodimer, where the D′D3 domains to bind FVIII and preventsFVIII interaction with phospholipids and activated protein C and/orpreventing or inhibiting binding to endogenous VWF. Protein frompSYN-FVIII-064 is expressed in the cell as a single polypeptide wherethe C-terminus of the FVIII-Fc subunit is linked to the N-terminus ofthe VWF D′D3-Fc subunit by a 6× (GGGGS) polypeptide linker (SEQ ID NO:74). In addition, RRRRS (SEQ ID NO: 75) and RKRRKR (SEQ ID NO: 76)sequences were inserted at the 5′ and 3′ end of the polypeptide linker,respectively, for intracellular cleavage by proprotein convertasesfollowing the last Arg at each sequence. Hence, the cells can express adouble chain FVIII-Fc/D′D3-Fc heterodimer where the FVIII-Fc chain has aRRRRS sequence (SEQ ID NO: 75) at the C-terminus, but the remainder ofthe linker sequence has been removed. Another 3× (GGGGS) polypeptidelinker (SEQ ID NO: 28) along with a thrombin cleavage site is introducedin between the VWF domains and the Fc region to facilitate release ofthe VWF fragment from FVIII once the FVIII-VWF hetero-dimeric protein isactivated by thrombin allowing interaction of FVIII with other clottingfactors.

Synthesis of the DNA fragments containing a portion of the first Fcregion followed by a 6× (GGGGS) (SEQ ID NO: 74), the VWF-D′D3 domain(1-477aa; C336A/C379A mutation), 3× (GGGGS) (SEQ ID NO:28), the thrombincleavage site and a portion of the second Fc was outsourced(Genscript-sequence number 103069, shown below). A fragment of Genscriptconstruct was sub cloned into the SalI/RsRII digested pSYN-FVIII-049,which is FVIII-Fc construct with a cleavable linker in between two Fcdomains.

Genscript-Sequence number 103069 (SEQ ID NO: 82):CCGTCGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAACGGCGCCGCCGGAGCGGTGGCGGCGGATCAGGTGGGGGTGGATCAGGCGGTGGAGGTTCCGGTGGCGGGGGATCCGGCGGTGGAGGTTCCGGTGGGGGTGGATCAAGGAAGAGGAGGAAGAGAAGCCTATCCTGTCGGCCCCCCATGGTCAAGCTGGTGTGTCCCGCTGACAACCTGCGGGCTGAAGGGCTCGAGTGTACCAAAACGTGCCAGAACTATGACCTGGAGTGCATGAGCATGGGCTGTGTCTCTGGCTGCCTCTGCCCCCCGGGCATGGTCCGGCATGAGAATCGATGTGTGGCCCTGGAAAGGTGTCCCTGCTTCCATCAGGGCAAGGAGTATGCCCCTGGAGAAACAGTGAAGATTGGCTGCAACACTTGTGTCTGTCGGGACCGGAAGTGGAACTGCACAGACCATGTGTGTGATGCCACGTGCTCCACGATCGGCATGGCCCACTACCTCACCTTCGACGGGCTCAAATACCTGTTCCCCGGGGAGTGCCAGTACGTTCTGGTGCAGGATTACTGCGGCAGTAACCCTGGGACCTTTCGGATCCTAGTGGGGAATAAGGGATGCAGCCACCCCTCAGTGAAATGCAAGAAACGGGTCACCATCCTGGTGGAGGGAGGAGAGATTGAGCTGTTTGACGGGGAGGTGAATGTGAAGAGGCCCATGAAGGATGAGACTCACTTTGAGGTGGTGGAGTCTGGCCGGTACATCATTCTGCTGCTGGGCAAAGCCCTCTCCGTGGTCTGGGACCGCCACCTGAGCATCTCCGTGGTCCTGAAGCAGACATACCAGGAGAAAGTGTGTGGCCTGTGTGGGAATTTTGATGGCATCCAGAACAATGACCTCACCAGCAGCAACCTCCAAGTGGAGGAAGACCCTGTGGACTTTGGGAACTCCTGGAAAGTGAGCTCGCAGTGTGCTGACACCAGAAAAGTGCCTCTGGACTCATCCCCTGCCACCTGCCATAACAACATCATGAAGCAGACGATGGTGGATTCCTCCTGTAGAATCCTTACCAGTGACGTCTTCCAGGACTGCAACAAGCTGGTGGACCCCGAGCCATATCTGGATGTCTGCATTTACGACACCTGCTCCTGTGAGTCCATTGGGGACTGCGCCGCATTCTGCGACACCATTGCTGCCTATGCCCACGTGTGTGCCCAGCATGGCAAGGTGGTGACCTGGAGGACGGCCACATTGTGCCCCCAGAGCTGCGAGGAGAGGAATCTCCGGGAGAACGGGTATGAGGCTGAGTGGCGCTATAACAGCTGTGCACCTGCCTGTCAAGTCACGTGTCAGCACCCTGAGCCACTGGCCTGCCCTGTGCAGTGTGTGGAGGGCTGCCATGCCCACTGCCCTCCAGGGAAAATCCTGGATGAGCTTTTGCAGACCTGCGTTGACCCTGAAGACTGTCCAGTGTGTGAGGTGGCTGGCCGGCGTTTTGCCTCAGGAAAGAAAGTCACCTTGAATCCCAGTGACCCTGAGCACTGCCAGATTTGCCACTGTGATGTTGTCAACCTCACCTGTGAAGCCTGCCAGGAGCCGATCGATGGCGGTGGAGGTTCCGGTGGCGGGGGATCCCTGGTCCCCCGGGGCAGCGGAGGCGACAAAACTCACACATGCCCACCGTGCCCAGCTCCAGAACTCCTGGGCGGACCGTCA

(b) Cloning of pSYN-FVIII-065

The FVIII-065 plasmid comprises the first 276 amino acids of the D′D3domain of VWF attached to a second Fc region. The VWF fragment was PCRamplified from full-length VWF plasmid pSYN-VWF-008 by using primercombinations ESC17 and ESC41.

ESC17-Fwd-VWF cloning oligo with ClaI (SEQ ID NO: 77)GTCCGGCATGAGAATCGATGTGTG ESC41-Rev-VWF with EcoRV (SEQ ID NO: 78)CCTCCACCGCCAGATATCAGAGGCACTTTTC

The expected sized band (-692 bp) was gel purified with a Gel Extractionkit (Qiagen, Valencia, Calif.) and cloned into the Cla1 and EcoRV sitesof pSYN-FVIII-064 to generate pSYN-FVIII-065.

Example 3: Cloning of pSYN-FVIII-159, 160, 178, 179 (FIG. 3)

In order to vary the linker length between the VWF fragment and Fcregion, an EcoRV site was introduced at the junction of VWF and thebeginning of 20 amino acid linker in pSYN-FVIII-064, variable sizelinkers were then used to replace the 20 aa linker in PSYN-FVIII-064.The new DNA constructs are: pSYN-FVIII-159, 160, 178, and 179 whichcontains 35 aa, 48 aa, 73 aa and 98 aa linkers, respectively.

To insert a 35 amino acid linker in pSYN-FVIII-159, two oligos (ESC78-105 bp and ESC79-107 bp) were ordered from Integrated DNA Technologies,Inc (Coralville, Iowa). Oligos were annealed and extended using astandard PCR method:

Primers: ESC78-Fwd with EcoRV site (SEQ ID NO: 79)AAAGTGCCTCTGATATCTGGCGGTGGAGGTTCCGGTGGCGGGGGATCCGGTGGCGGGGGATCCGGTGGCGGGGGATCCGGTGGCGGGGGATCCCTGGT CCCCCGGESC79-Rev with RsRII site (SEQ ID NO: 80)GAAGAGGAAGACTGACGGTCCGCCCAGGAGTTCTGGAGCTGGGCACGGTGGGCATGTGTGAGTTTTGTCGCCTCCGCTGCCCCGGGGGACCAGGGATC CCCCGCCAC

A 50 μl PCR oligo annealing and extension reaction was carried out withESC78/ESC79 primer combo using the 3 step PCR amplification cycle: 25cycles of (96° C. 30 seconds, 55° C. 30 seconds, 68° C. 30 seconds). Theexpected sized band (-186 bp) was gel purified with a Gel Extraction kit(Qiagen, Valencia, Calif.) and cloned into the EcoRV and RsRIIrestriction sites of pSYN-FVIII-064 to generate pSYN-FVIII-159.

(b) Cloning pSYN-FVIII-160, 178, and 179

pSYN-VIII-160 has a 48 amino acids linker in between the VWF fragmentand the Fc region. Synthesis of DNA fragment coding for 48 amino acidslinker (ISGG GGSGGGGSGGGGSGGGGSGGGGSGGGGSLVPRGSGGGGSGGGGS) (SEQ ID NO:81) and a portion of the Fc region was outsourced (Genscript-Sequenceno-132601, shown below). A fragment of the Genscript construct was subcloned into the EcoRV/RsRII digested pSYN-FVIII-0159 (mentioned above).

Genscript-Sequence no-132601 (SEQ ID NO: 83)AAAGTGCCTCTGATATCTGGCGGTGGAGGTTCCGGTGGCGGGGGATCCGGCGGTGGAGGTTCCGGCGGTGGAGGTTCCGGTGGCGGGGGATCCGGTGGCGGGGGATCCCTGGTCCCCCGGGGCAGCGGCGGTGGAGGTTCCGGTGGCGGGGGATCCGACAAAACTCACACATGCCCACCGTGCCCAGCTCCAGAACTCCTGGGCGGACCGTCAGTCTTCC

pSYN-VIII-178 has a 73 amino acids linker in between the VWF fragmentand the Fc region. Synthesis of DNA fragment coding for 73 amino acidslinker (|SGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSLVPRGSGGGGSGGGGS) (SEQ ID NO: 84) and a portion of Fc region was outsourced(Genscript-Sequence no-144849, shown below). A fragment of Genscriptconstruct was sub cloned into the EcoRV/RsRII digested pSYN-FVIII-0159(mentioned above).

Genscript-Sequence #-144849 (SEQ ID NO: 85)GCCTGCCAGGAGCCGATATCTGGCGGTGGAGGTTCCGGTGGCGGGGGATCCGGCGGTGGAGGTTCCGGCGGTGGAGGTTCCGGTGGCGGGGGATCCGGCGGTGGAGGTTCCGGTGGCGGGGGATCCGGCGGTGGAGGTTCCGGCGGTGGAGGTTCCGGTGGCGGGGGATCCGGTGGCGGGGGATCCCTGGTCCCCCGGGGCAGCGGCGGTGGAGGTTCCGGTGGCGGGGGATCCGACAAAACTCACACATGCCCCCGTGCCCAGCTCCAGAACTCCTGGGCGGACCGTCAGTCT TCCTC

pSYN-VIII-179 has a 98 amino acids linker in between the VWF fragmentand the Fc region. Synthesis of DNA fragment coding for 98 amino acidslinker (ISGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSLVPRGSGGGGSGGGGS) (SEQ ID NO: 86) and a portionof Fc region was outsourced (Genscript-Sequence no-144849 shown below).A fragment of Genscript construct was sub cloned into the EcoRV/RsRIIdigested pSYN-FVIII-0159 (mentioned above).

Genscript-Sequence #-144849 (SEQ ID NO: 87)GCCTGCCAGGAGCCGATATCTGGCGGTGGAGGTTCCGGTGGCGGGGGATCCGGCGGTGGAGGTTCCGGCGGTGGAGGTTCCGGTGGCGGGGGATCCGGCGGTGGAGGTTCCGGTGGCGGGGGATCCGGCGGTGGAGGTTCCGGCGGTGGAGGTTCCGGTGGCGGGGGATCCGGCGGTGGAGGTTCCGGTGGCGGGGGATCCGGCGGTGGAGGTTCCGGCGGTGGAGGTTCCGGTGGCGGGGGATCCGGTGGCGGGGGATCCCTGGTCCCCCGGGGCAGCGGCGGTGGAGGTTCCGGTGGCGGGGGATCCGACAAAACTCACACATGCCCACCGTGCCCAGCTCCAGAACTCCTGGGCGGA CCGTCAGTCTTCCTCTTCCCCloning of pSYN-FVIII-180, 181, and 182

pSYN-FVIII-180, 181, and 182 were constructed from pSYN-FVIII-160.K2093A or F2093A or K2093A/F2093A mutations were introduced into the C1domain of FVIII in pSYN-FVIII-160 to form pSYN-FVIII-180, pSYN-FVIII-181and pSYN-FVIII-182 respectively.

FVIII-VWF-Fc Heterodimer Protein Sequence (SEQ ID NO: 88)

(FVIII sequence amino acid position 1-1457; underlined region representsFc region; curvy underline represents cleavable linker in between firstFc and VWF fragment; double underlined region represents VWF fragment;bold region represents variable length cleavable linker in between VWFfragment and Fc. The linker length varies in FVIII-064, 159, 160, 178,and 179 constructs).

1 MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQSDL GELPVDARFP 51PRVPKSFPFN TSVVYKKTLF VEFTDHLFNI AKPRPPWMGL LGPTIQAEVY 101DTVVITLKNM ASHPVSLHAV GVSYWKASEG AEYDDQTSQR EKEDDKVFPG 151GSHTYVWQVL KENGPMASDP LCLTYSYLSH VDLVKDLNSG LIGALLVCRE 201GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD AASARAWPKM 251HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL EGHTFLVRNH 301RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME AYVKVDSCPE 351EPQLRMKNNE EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI RSVAKKHPKT 401WVHYIAAEEE DWDYAPLVLA PDDRSYKSQY LNNGPQRIGR KYKKVRFMAY 451TDETFKTREA IQHESGILGP LLYGEVGDTL LIIFKNQASR PYNIYPHGIT 501DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP TKSDPRCLTR 551YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR NVILFSVFDE 601NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHSINGYV FDSLQLSVCL 651HEVAYWYILS IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF PFSGETVFMS 701MENPGLWILG CHNSDFRNRG MTALLKVSSC DKNTGDYYED SYEDISAYLL 751SKNNAIEPRS FSQNPPVLKR HQREITRTTL QSDQEEIDYD DTISVEMKKE 801DFDIYDEDEN QSPRSFQKKT RHYFIAAVER LWDYGMSSSP HVLRNRAQSG 851SVPQFKKVVF QEFTDGSFTQ PLYRGELNEH LGLLGPYIRA EVEDNIMVTF 901RNQASRPYSF YSSLISYEED QRQGAEPRKN FVKPNETKTY FWKVQHHMAP 9511TKDEFDCKAW AYFSDVDLEK DVHSGLIGPL LVCHTNTLNP AHGRQVTVQE 1001FALFFTIFDE TKSWYFTENM ERNCRAPCNI QMEDPTFKEN YRFHAINGYI 1051MDTLPGLVMA QDQRIRWYLL SMGSNENIHS IHFSGHVFTV RKKEEYKMAL 1101YNLYPGVFET VEMLPSKAGI WRVECLIGEH LHAGMSTLFL VYSNKCQTPL 1151GMASGHIRDF QITASGQYGQ WAPKLARLHY SGSINAWSTK EPFSWIKVDL 1201LAPMIIHGIK TQGARQKFSS LYISQFIIMY SLDGKKWQTY RGNSTGTLMV 1251FFGNVDSSGI KHNIFNPPII ARYIRLHPTH YSIRSTLRME LMGCDLNSCS 1301MPLGMESKAI SDAQITASSY FTNMFATWSP SKARLHLQGR SNAWRPQVNN 1351PKEWLQVDFQ KTMKVTGVTT QGVKSLLTSM YVKEFLISSS QDGHQWTLFF 1101QNGKVKVFQG NQDSFTPVVN SLDPPLLTRY LRIHPQSWVH QIALRMEVLG 1451CEAQDLYDKT HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV 1501VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV SVLTVLHQDW 1551LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP SRDELTKNQV 1601SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD 1651KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGKR

1701

SLSCR PPMVKLVCPA DNLRAEGLEC 1751TKTCQNYDLE CMSMGCVSGC LCPPGMVRHE NRCVALERCP CFHQGKEYAP 1801GETVKIGCNT CVCRDRKWNC TDHVCDATCS TIGMAHYLTF DGLKYLFPGE 1851CQYVLVQDYC GSNPGTFRIL VGNKGCSHPS VKCKKRVTIL VEGGEIELFD 1901GEVNVKRPMK DETHFEVVES GRYIILLLGK ALSVVWDRHL SISVVLKQTY 1051QEKVCGLCGN FDGIQNNDLT SSNLQVEEDP VDFGNSWKVS SQCADTRKVP 2001LDSSPATCHN NIMKQTMVDS SCRILTSDVF QDCNKLVDPE PYLDVCIYDT 2051CSCESIGDCA AFCDTIAAYA HVCAQHGKVV TWRTATLCPQ SCEERNLREN 2101GYEAEWRYNS CAPACQVTCQ HPEPLACPVQ CVEGCHAHCP PGKILDELLQ 2151TCVDPEDCPV CEVAGRRFAS GKKVTLNPSD PEHCQICHCD VVNLTCEACQ 2201 EPIDGGGGSG GGGSLVPRGS GG DKTHTCPP CPAPELLGGP SVFLFPPKPK 2251DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS 2301TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV 2351YTLPPSRDEL TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL 2401DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGK

Example 4: Example of FVIII-VWF DNA Constructs (FIG. 4)

The VWF fragment and FVIII protein can be linked together by a linker oranother protein or a polypeptide using conventional recombinant DNAtechniques, as show in FIG. 4 . In FIG. 4A, the D1D2D′D3 domains of VWFis linked to the FVIII protein by a 48aalinker-ISGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSLVPRGSGGGGSGGGGS (SEQ ID NO: 89)and protects FVIII from premature clearance. To further enhance theFVIII protecting activity of D′D3, another protein or polypeptide thathas half-life extension potential such as albumin or a PAS sequence(heterologous moieties) can be incorporated into the construct. Theheterologous moiety, e.g., albumin protein or PAS sequence, can beincorporated into different positions of the FVIII molecule; a fewexamples were shown in FIG. 4B-4D: at the N-termini of FVIII (4B), atthe C-termini of FVIII (4C), or in the B region (4D). In thoseconstructs, the additional protein sequences could enhance the D′D3protecting activity and further extend FVIII half-life.

In addition, a heterologous moiety, e.g., albumin or PAS sequence, canalso be incorporated into the FVIII/VWF heterodimer constructs as shownin FIG. 4E-4G. In FIG. 4E, a heterologous moiety, e.g., albumin or PASsequence, is incorporated into the FVIII B domain region of FVIII-148;In FIG. 4F, a heterologous moiety, e.g., albumin or PAS sequence, isincorporated into the FVIII B domain region of FVIII-136; In FIG. 4G, aheterologous moiety, e.g., albumin or PAS sequence, is used as a linkerto connect D′D3 fragment and Fc. In those configurations, a synergeticeffect of D′D3, Fc, and heterologous moiety that is a half-life extender(e.g., albumin/PAS sequence) is expected on FVIII half-life extension.

Example 5: Plasmid Construction of Co-Transfection System forFVIIIFc-VWF Heterodimer (FIG. 5)

A co-transfection system was generated for FVIIIFc-VWF heterodimerproduction, which contains three DNA constructs. The first DNAconstruct-pSYN-FVIII-155 is encoding a FVIII-Fc fusion protein in whicha single chain FVIII protein was directly fused to a single Fc fragment,and the second DNA construct is pSYN-VWF-031, which encodes a D′D3-Fcfusion protein (mentioned above in example 1). HEK293F cells weretransfected with the two plasmids along with a third plasmid (PC5) at a80:15:5 ratio. Co-transfection with PC5 is to ensure full propeptideprocessing of the D1 and D2 regions so that we have mature D′D3 domains.The synthesized proteins were secreted as FVIIIFc/D′D3Fc heterodimer andD′D3Fc homodimer and the FVIIIFc/D′D3Fc heterodimer was separated fromthe D′D3Fc homodimer by protein purification.

pSYN-FVIII-155 mature Protein sequencing (SEQ ID NO: 90):ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDWRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKAHQAEITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGKpSYN-FVIII-155 DNA sequencing (SEQ ID NO: 91):ATGCAAATAGAGCTCTCCACCTGCTTCTTTCTGTGCCTTTTGCGATTCTGCTTTAGTGCCACCAGAAGATACTACCTGGGTGCAGTGGAACTGTCATGGGACTATATGCAAAGTGATCTCGGTGAGCTGCCTGTGGACGCAAGATTTCCTCCTAGAGTGCCAAAATCTTTTCCATTCAACACCTCAGTCGTGTACAAAAAGACTCTGTTTGTAGAATTCACGGATCACCTTTTCAACATCGCTAAGCCAAGGCCACCCTGGATGGGTCTGCTAGGTCCTACCATCCAGGCTGAGGTTTATGATACAGTGGTCATTACACTTAAGAACATGGCTTCCCATCCTGTCAGTCTTCATGCTGTTGGTGTATCCTACTGGAAAGCTTCTGAGGGAGCTGAATATGATGATCAGACCAGTCAAAGGGAGAAAGAAGATGATAAAGTCTTCCCTGGTGGAAGCCATACATATGTCTGGCAGGTCCTGAAAGAGAATGGTCCAATGGCCTCTGACCCACTGTGCCTTACCTACTCATATCTTTCTCATGTGGACCTGGTAAAAGACTTGAATTCAGGCCTCATTGGAGCCCTACTAGTATGTAGAGAAGGGAGTCTGGCCAAGGAAAAGACACAGACCTTGCACAAATTTATACTACTTTTTGCTGTATTTGATGAAGGGAAAAGTTGGCACTCAGAAACAAAGAACTCCTTGATGCAGGATAGGGATGCTGCATCTGCTCGGGCCTGGCCTAAAATGCACACAGTCAATGGTTATGTAAACAGGTCTCTGCCAGGTCTGATTGGATGCCACAGGAAATCAGTCTATTGGCATGTGATTGGAATGGGCACCACTCCTGAAGTGCACTCAATATTCCTCGAAGGTCACACATTTCTTGTGAGGAACCATCGCCAGGCGTCCTTGGAAATCTCGCCAATAACTTTCCTTACTGCTCAAACACTCTTGATGGACCTTGGACAGTTTCTACTGTTTTGTCATATCTCTTCCCACCAACATGATGGCATGGAAGCTTATGTCAAAGTAGACAGCTGTCCAGAGGAACCCCAACTACGAATGAAAAATAATGAAGAAGCGGAAGACTATGATGATGATCTTACTGATTCTGAAATGGATGTGGTCAGGTTTGATGATGACAACTCTCCTTCCTTTATCCAAATTCGCTCAGTTGCCAAGAAGCATCCTAAAACTTGGGTACATTACATTGCTGCTGAAGAGGAGGACTGGGACTATGCTCCCTTAGTCCTCGCCCCCGATGACAGAAGTTATAAAAGTCAATATTTGAACAATGGCCCTCAGCGGATTGGTAGGAAGTACAAAAAAGTCCGATTTATGGCATACACAGATGAAACCTTTAAGACTCGTGAAGCTATTCAGCATGAATCAGGAATCTTGGGACCTTTACTTTATGGGGAAGTTGGAGACACACTGTTGATTATATTTAAGAATCAAGCAAGCAGACCATATAACATCTACCCTCACGGAATCACTGATGTCCGTCCTTTGTATTCAAGGAGATTACCAAAAGGTGTAAAACATTTGAAGGATTTTCCAATTCTGCCAGGAGAAATATTCAAATATAAATGGACAGTGACTGTAGAAGATGGGCCAACTAAATCAGATCCTCGGTGCCTGACCCGCTATTACTCTAGTTTCGTTAATATGGAGAGAGATCTAGCTTCAGGACTCATTGGCCCTCTCCTCATCTGCTACAAAGAATCTGTAGATCAAAGAGGAAACCAGATAATGTCAGACAAGAGGAATGTCATCCTGTTTTCTGTATTTGATGAGAACCGAAGCTGGTACCTCACAGAGAATATACAACGCTTTCTCCCCAATCCAGCTGGAGTGCAGCTTGAGGATCCAGAGTTCCAAGCCTCCAACATCATGCACAGCATCAATGGCTATGTTTTTGATAGTTTGCAGTTGTCAGTTTGTTTGCATGAGGTGGCATACTGGTACATTCTAAGCATTGGAGCACAGACTGACTTCCTTTCTGTCTTCTTCTCTGGATATACCTTCAAACACAAAATGGTCTATGAAGACACACTCACCCTATTCCCATTCTCAGGAGAAACTGTCTTCATGTCGATGGAAAACCCAGGTCTATGGATTCTGGGGTGCCACAACTCAGACTTTCGGAACAGAGGCATGACCGCCTTACTGAAGGTTTCTAGTTGTGACAAGAACACTGGTGATTATTACGAGGACAGTTATGAAGATATTTCAGCATACTTGCTGAGTAAAAACAATGCCATTGAACCAAGAAGCTTCTCTCAAAACCCACCAGTCTTGAAAGCCCATCAGGCGGAAATAACTCGTACTACTCTTCAGTCAGATCAAGAGGAAATTGACTATGATGATACCATATCAGTTGAAATGAAGAAGGAAGATTTTGACATTTATGATGAGGATGAAAATCAGAGCCCCCGCAGCTTTCAAAAGAAAACACGACACTATTTTATTGCTGCAGTGGAGAGGCTCTGGGATTATGGGATGAGTAGCTCCCCACATGTTCTAAGAAACAGGGCTCAGAGTGGCAGTGTCCCTCAGTTCAAGAAAGTTGTTTTCCAGGAATTTACTGATGGCTCCTTTACTCAGCCCTTATACCGTGGAGAACTAAATGAACATTTGGGACTCCTGGGGCCATATATAAGAGCAGAAGTTGAAGATAATATCATGGTAACTTTCAGAAATCAGGCCTCTCGTCCCTATTCCTTCTATTCTAGCCTTATTTCTTATGAGGAAGATCAGAGGCAAGGAGCAGAACCTAGAAAAAACTTTGTCAAGCCTAATGAAACCAAAACTTACTTTTGGAAAGTGCAACATCATATGGCACCCACTAAAGATGAGTTTGACTGCAAAGCCTGGGCTTATTTCTCTGATGTTGACCTGGAAAAAGATGTGCACTCAGGCCTGATTGGACCCCTTCTGGTCTGCCACACTAACACACTGAACCCTGCTCATGGGAGACAAGTGACAGTACAGGAATTTGCTCTGTTTTTCACCATCTTTGATGAGACCAAAAGCTGGTACTTCACTGAAAATATGGAAAGAAACTGCAGGGCTCCCTGCAATATCCAGATGGAAGATCCCACTTTTAAAGAGAATTATCGCTTCCATGCAATCAATGGCTACATAATGGATACACTACCTGGCTTAGTAATGGCTCAGGATCAAAGGATTCGATGGTATCTGCTCAGCATGGGCAGCAATGAAAACATCCATTCTATTCATTTCAGTGGACATGTGTTCACTGTACGAAAAAAAGAGGAGTATAAAATGGCACTGTACAATCTCTATCCAGGTGTTTTTGAGACAGTGGAAATGTTACCATCCAAAGCTGGAATTTGGCGGGTGGAATGCCTTATTGGCGAGCATCTACATGCTGGGATGAGCACACTTTTTCTGGTGTACAGCAATAAGTGTCAGACTCCCCTGGGAATGGCTTCTGGACACATTAGAGATTTTCAGATTACAGCTTCAGGACAATATGGACAGTGGGCCCCAAAGCTGGCCAGACTTCATTATTCCGGATCAATCAATGCCTGGAGCACCAAGGAGCCCTTTTCTTGGATCAAGGTGGATCTGTTGGCACCAATGATTATTCACGGCATCAAGACCCAGGGTGCCCGTCAGAAGTTCTCCAGCCTCTACATCTCTCAGTTTATCATCATGTATAGTCTTGATGGGAAGAAGTGGCAGACTTATCGAGGAAATTCCACTGGAACCTTAATGGTCTTCTTTGGCAATGTGGATTCATCTGGGATAAAACACAATATTTTTAACCCTCCAATTATTGCTCGATACATCCGTTTGCACCCAACTCATTATAGCATTCGCAGCACTCTTCGCATGGAGTTGATGGGCTGTGATTTAAATAGTTGCAGCATGCCATTGGGAATGGAGAGTAAAGCAATATCAGATGCACAGATTACTGCTTCATCCTACTTTACCAATATGTTTGCCACCTGGTCTCCTTCAAAAGCTCGACTTCACCTCCAAGGGAGGAGTAATGCCTGGAGACCTCAGGTGAATAATCCAAAAGAGTGGCTGCAAGTGGACTTCCAGAAGACAATGAAAGTCACAGGAGTAACTACTCAGGGAGTAAAATCTCTGCTTACCAGCATGTATGTGAAGGAGTTCCTCATCTCCAGCAGTCAAGATGGCCATCAGTGGACTCTCTTTTTTCAGAATGGCAAAGTAAAGGTTTTTCAGGGAAATCAAGACTCCTTCACACCTGTGGTGAACTCTCTAGACCCACCGTTACTGACTCGCTACCTTCGAATTCACCCCCAGAGTTGGGTGCACCAGATTGCCCTGAGGATGGAGGTTCTGGGCTGCGAGGCACAGGACCTCTACGACAAAACTCACACATGCCCACCGTGCCCAGCTCCAGAACTCCTGGGCGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGTTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTG TCTCCGGGTAAA

Additional VWF fragments and FVIIIFC-VWF heterodimers that have beenconstructed are listed below.

TABLE 6 VWF Fragments and FVIII/VWF Heterodimer Constructs ConstructDescription Vector VWF pSYN-VWF-001 FVIII signal peptide D′D3 region(1-276 amino acids long 6x pcDNA 4 His) pSYN-VWF-002 FVIII signalpeptide D′D3 region (1-477 amino acids long 6x pcDNA 4 His) pSYN-VWF-003FVIII signal peptide D′D3 region partial A1 (1-511 amino acids pcDNA 4long 6x His) pSYN-VWF-004 FVIII signal peptide D′D3A1 region (1-716amino acids long 6x pcDNA 4 His) pSYN-VWF-006 D1D2D′D3-linker-CK1 pcDNA3.1 pSYN-VWF-008 Full length WT- VWF pcDNA 3.1 pSYN-VWF-009 D1D2D′D3region (1-276 aa, 6x His) pcDNA 3.1 pSYN-VWF-010 D1D2D′D3 region (1-477aa, 6x His) pcDNA 3.1 pSYN-VWF-011 D1D2D′D3 region partial A1 (1-511 aa,6x His) pcDNA 3.1 pSYN-VWF-012 D1D2D′D3A1 region (1-716 aa, 6x His)pcDNA 3.1 pSYN-VWF-013 D1D2D′D3 region (1-477 aa, C336A/C379A, 6x His)pcDNA 3.1 pSYN-VWF-014 FVIII signal peptide-D′D3 (1-477aa,C336A/C379A)-single Fc pcDNA 4 with 20aa linker containing thrombin sitepSYN-VWF-015 D1D2D′D3 (1-477aa, C336A/C379A)-single Fc with 20aa linkerpcDNA 4 containing thrombin site pSYN-VWF-016 FVIII signal peptide-D′D3(1-477aa, WT)-single Fc with 20aa pcDNA 4 linker containing thrombinsite pSYN-VWF-017 D1D2D′D3 (1-477aa, WT)-single Fc with 20aa linkercontaining pcDNA 4 thrombin site pSYN-VWF-025 D1D2D′D3 region (1-477 aa,6x His) in pLIVE pLIVE pSYN-VWF-029 D1D2D′D3 region (1-477 aa,C336A/C379A, 6x His) in pLIVE pLIVE pSYN-VWF-030 FVIII signalpeptide-D′D3 (l-477aa, C336A/C379A)-single Fc pcDNA 4 with 48aa linkercontaining thrombin site pSYN-VWF-031 D1D2D′D3 (1-477aa,C336A/C379A)-single Fc with 48aa linker pcDNA 4 containing thrombin sitepSYN-VWF-032 FVIII signal peptide-D′D3 (1-477aa, WT)-single Fc with 48aapcDNA 4 linker containing thrombin site pSYN-VWF-033 FVIII signalpeptide-D′D3 (1-477aa, WT)-single Fc with 35 aa pcDNA 4 linkercontaining thrombin site FVIII pSYN-FVIII-055 BDD-FVIII scFc with R336Iand Y1680F pBUD pSYN-FVIII-056 BDD-FVIII scFc with R336I, R562 andY1680F pBUD pSYN-FVIII-057 BDD-FVIII scFc with Y1680F pBUDpSYN-FVIII-058 BDD-FVIII scFc with S488A pBUD pSYN-FVIII-059 BDD-FVIIIscFc with R336I, R562K, S488A pBUD pSYN-FVIII-060 BDD-FVIII scFc withR336I, R562K, Y1680F pBUD pSYN-FVIII-061 BDD-FVIII scFc with R336I,R562K, S488A, Y1680F pBUD pSYN-FVIII-064 BDD-FVIII cleavable scFc withVWF D′D3 (1-477aa, pBUD C336A/C379A) on second Fc & 20aa thrombincleavable linker in between pSYN-FVIII-065 BDD-FVIII cleavable scFc withVWF D′D3 (1-276aa) on pBUD second Fc & 20aa thrombin cleavable linker inbetween pSYN-FVIII-083 BDD-FVIII scFc with R336I, S488A, R562K, Y1680F,E1984V pBUD pSYN-FVIII-086 BDD-FVIII scFc with 6x(GGGGS) linker inbetween C2 of pBUD FVIII and Fc pSYN-FVIII-095 BDD-FVIII scFc withS104C, R562K, Y1680F, G1960C pBUD pSYN-FVIII-101 BDD-FVIII scFc fromFVIII-041 into pcDNA 3.3. Topo pcDNA 3.3 Topo pSYN-FVIII-102 BDD-FVIII(M662C/D1828C for disulfide binding; APC pBUD cleavage mutationsR336I/R562K; along with Y1680F mutation for VWF binding) pSYN-FVIII-103BDD-FVIII scFc (Y662C/T1828C) pBUD pSYN-FVIII-104 BDD-FVIII scFc(G655C/ST1788C) pBUD pSYN-FVIII-113 BDD-FVIII (R490A/H497A) cleavablescFc with VWF D′D3 pBUD (1-477aa, C336A/C379A) on second Fc & 20aathrombin cleavable linker in between pSYN-FVIII-114 BDD-FVIII(R490A/H497A) cleavable scFc with VWF D′D3 pBUD (1-276) on second Fc &20aa thrombin cleavable linker in between pSYN-FVIII-126 BDD-FVIII scFc(M662C/D1828C) pcDNA 3.3 Topo pSYN-FVIII-127 BDD-FVIII scFc(M662C/D1828C for disulfide binding; APC pcDNA 3.3 cleavage mutationsR336I/R562K; along with Y1680F mutation Topo for VWF binding)pSYN-FVIII-128 BDD-FVIII scFc (Y664C/T1826C) pcDNA 3.3 TopopSYN-FVIII-129 mutation of R336I R562K R490A H497A N1224A in the pBUDbackground of pSYN-VIII-64 pSYN-FVIII-130 mutation of R336I R562K R490AH497A N1224A in the pBUD background of pSYN-VIII-65 pSYN-FVIII-131mutation of R471A Y487A R490A H497A N1224A in the pBUD background ofpSYN-VIII-64 pSYN-FVIII-132 mutation of R471A Y487A R490A H497A N1224Ain the pBUD background of pSYN-VIII-65 pSYN-FVIII-135 BDD- FVIII scFcwith R1645A/R1648A pcDNA 3.3 Topo pSYN-FVIII-136 BDD-FVIII cleavablescFc with VWF D′D3 (1-477aa, pcDNA 3.3 C336A/C379A) on second Fc & 20aathrombin cleavable linker Topo in between pSYN-FVIII-137 BDD-FVIIIcleavable scFc with VWF D′D3 (1-276aa) on pcDNA 3.3 second Fc & 20aathrombin cleavable linker in between Topo pSYN-FVIII-145 BDD- FVIII scFcwith R471A/Y487A, R490A/H497A pcDNA 3.3 Topo pSYN-FVIII-146 BDD- FVIIIcleavable scFc (R471A/Y487A) with VWF D′D3 pcDNA 3.3 (1-477aa,C336A/C379A) on second Fc & 20aa thrombin Topo cleavable linkerpSYN-FVIII-147 BDD- FVIII cleavable scFc (R471A/Y487A) with VWF D′D3pcDNA 3.3 (1-276aa) on second Fc & 20aa thrombin cleavable linker inTopo between pSYN-FVIII-148 BDD- FVIII cleavable scFc (R1645A/R1648A)with VWF D′D3 pcDNA 3.3 (1-477aa, C336A/C379A) on second Fc & 20aathrombin Topo cleavable linker pSYN-FVIII-149 BDD- FVIII cleavable scFc(R1645A/R1648A) with VWF D′D3 pcDNA 3.3 (1-276aa) on second Fc & 20aathrombin cleavable linker Topo pSYN-FVIII-155 BDD- FVIII fused to singleFc (R1645A/R1648A) pcDNA 4 pSYN-FVIII-159 BDD-FVIII cleavable scFc withVWF D′D3 (1-477aa, pBUD C336A/C379A) on second Fc & 35 aa thrombincleavable linker in between pSYN-FVIII-160 BDD-FVIII cleavable scFc withVWF D′D3 (1-477aa, pBUD C336A/C379A) on second Fc & 48 aa thrombincleavable linker in between pSYN-FVIII-164 BDD- FVIII cleavable scFc(R490A/H497A, R1645A/R1648A) pcDNA 3.3 with VWF D′D3 (1-477aa,C336A/C379A) on second Fc & Topo 20aa thrombin cleavable linkerpSYN-FVIII-165 BDD- FVIII cleavable scFc (R336I/R562K, R490A/H497A,pcDNA 3.3 R1645A/R1648A) with VWF D′D3 (1-477aa, C336A/C379A) Topo onsecond Fc & 20aa thrombin cleavable linker pSYN-FVIII-178 BDD-FVIIIcleavable scFc with VWF D′D3 (1-477aa, pBUD C336A/C379A) on second Fc &73 aa thrombin cleavable linker in between pSYN-FVIII-179 BDD-FVIIIcleavable scFc with VWF D′D3 (1-477aa, pBUD C336A/C379A) on second Fc &98 aa thrombin cleavable linker in between pSYN-FVIII-180 BDD-FVIII(K2092A) cleavable scFc with VWF D′D3 pBUD (1-477aa, C336A/C379A) onsecond Fc & 48 aa thrombin cleavable linker in between pSYN-FVIII-181BDD-FVIII (F2093A) cleavable scFc with VWF D′D3 pBUD (1-477aa,C336A/C379A) on second Fc & 48 aa thrombin cleavable linker in betweenpSYN-FVIII-182 BDD-FVIII (K2092A/F2093A) cleavable scFc with VWF D′D3pBUD (1-477aa, C336A/C379A) on second Fc & 48 aa thrombin cleavablelinker in between

Example 6: Protein Purification Protein Purification of VWF Fragments

The VWF fragments were purified through a two-step purification method.A Nickel Sulfate charged IMAC (Immobilized Metal AffinityChromatography) column was used for the primary purification, aFractogel DEAE ion exchange column was used for the final purification.The detail purification method is described below.

(a) Primary Purification of VWF Fragment on Nickel IMAC

A 14 mL Nickel IMAC Sepharose HP column [XK26/3] was equilibrated with25 mM HEPES, 500 mM NaCl, 10 mM Imidazole, and 0.05% Tween-20 @ pH 7.5.Approximately 7.2 L of VWF conditioned media was adjusted with 100 mL of1M HEPES @ pH 7.5 and 600 mL of 5M NaCl. Then 80 mL of 1M Imidazole (@pH 7.5) was added to a final concentration of 10 mM. The 7.8 L of theadjusted VWF conditioned media was then loaded onto the column at 2-8°C. at 10 mL/min [113 cm/hour]. The wash steps were performed at 13.3mL/minute [150 cm/hour]. First, a 2×Column Volume (CV) wash wasperformed with 25 mM HEPES, 500 mM NaCl, 10 mM Imidazole, and 0.05%Tween-20 @ pH 7.5 in normal flow {“DownFlow” }. Next, a 3×CV wash wasperformed with 25 mM HEPES, 500 mM NaCl, 10 mM Imidazole, and 0.05%Tween-20 @ pH 7.5 in reverse flow {“UpFlow”}. Lastly, A 3×CV wash wasperformed with 25 mM HEPES, 500 mM NaCl, 10 mM Imidazole, and 0.05%Tween-20 @ pH 7.5 in normal flow {“DownFlow”}. The elution was performedas a 10×CV gradient to 50% B1 (25 mM HEPES, 500 mM NaCl, 500 mMImidazole, and 0.05% Tween-20 @ pH 7.5). The fraction volume was set to10 mL. Then, the column was stripped with 100% B1. This was followed bya wash with 25 mM HEPES, 500 mM NaCl, 10 mM Imidazole, and 0.05%Tween-20 @ pH 7.5. A second Strip was performed with 1N NaOH. Then thecolumn was flushed with 1M TRIS, 1M NaCl @ pH 7.8, followed by 25 mMHEPES, 500 mM NaCl, 10 mM Imidazole, and 0.05% Tween-20 @ pH 7.5.Finally, the column was flushed with 5 CV's of DPBS+20% Ethanol andstored at 4° C.

(b) Secondary Purification of VWF Fragment on Fractogel DEAE

Secondary purification of VWF fragment was performed on Fractogel DEAE @pH 7.5. Firstly, 20 mL of VWF Nickel IMAC eluate (corresponding to theVWF fragment peak) was adjusted with 200 mg of Zwittergent 3-14zwitterionic detergent in an attempt to disrupt aggregated specieswithout using denaturing or reducing excipients. After the detergent wasdissolved, the protein was left at RT for approximately 15 minutes.Then, the protein was adjusted with 4 grams of trehalose, 1 mL of 10%Tween-20, 5 mL of 1M HEPES @ pH 7.5 and 174 mL of “Milli-Q” water. Theequilibration buffer “A12” was 25 mM HEPES, 50 mM NaCl, 1% Trehalose,0.05% Tween-20 @ pH 7.5. The elution buffer “B1” was 25 mM HEPES, 1000mM NaCl, 1% Trehalose, 0.05% Tween-20 @ pH 7.5. The elution wasperformed as a 10 CV gradient to 50% B1, with a 5+CV hold followed by astep to 100% B1. Then the column was stripped with 0.85% PhosphoricAcid, followed by 1M TRIS, 1M NaCl @ pH 7.5. Then the column wasstripped with 1N NaOH, 2M NaCl followed by 1M TRIS, 1M NaCl @ pH 7.5.Then the column was flushed with 25 mM HEPES, 100 mM NaCl+20% Ethanol @pH 7.5 for storage.

(c) Protein Purification of FVIII-VWF Heterodimer

The FVIII-VWF heterodimer was first purified by an affinity column (GEVIIISelect), then followed by a Fractogal TMAE ion exchange column.(McCue J T, Selvitelli K, Walker J, J Chromatogr A. 2009 Nov. 6;1216(45):7824-30. Epub 2009 Sep. 23.)

For the purification of FVIII-155/VWF-31, a tangential flow filtration(TFF) step was used to buffer exchange the clarified conditioned media.The targeted proteins in the filtrate were then captured using affinitychromatography. A weak anion exchange chromatography step was followedto reduce HMW species. Both the purity and size of the molecule wereaccessed by HPLC-SEC and SDS-PAGE. The presence of different domains ofFVIII-155/VWF-31 was further confirmed by western blotting. The specificactivity of the molecule was comparable to B-domain deleted FVIII.

(d) Thrombin Digestion of FVIII-VWF Heterodimer (FIG. 8 )

FVIII-VWF-Fc heterodimer or FVIII-Fc (control) was mixed with thrombinin 1:10 ratio in thrombin cleavage buffer (50 mM Tris, pH 7.4, 150 mMNaCl, 2 mM CaCl2, 5% Glycerol). The reaction was incubated at 37° C. for20 minutes. Digested product was run on 4-12% reducing tris-glycine gel.Undigested protein was used as a control. Bands were visualized bycoomassie stain.

(e) Evaluation of the VWF Binding Ability of FVIII-155/VWF-031 by OctetAssay

The VWF binding ability of FVIII-155/VWF-031 was determined by Bio-LayerInterferometry (BLI) based measurements (Octet assay) at 25° C. with aForteBio Octet 384 instrument using Tris binding buffer (50 mM Tris, pH7.2, 150 mM NaCl, 5 mM CaCl₂)). The Octet assay for determining FVIIIbinding was based on the hydrophobic immobilization of human vonWillebrand Factor (hVWF) (Haematologic Technologies Catalog No.HCVWF-0191) onto the APS Biosensor, followed by binding of 1.0% BovineSerum Albumin (Jackson ImmunoResearch Catalog No. 001-000-161). Briefly,hVWF (38.5 nM) was diluted in Tris buffer and loaded across APSBiosensors for 600 sec, yielding approximately 3.0-3.5 nm binding on thereaction probes. Control APS probes were loaded with 1.0% BSA in theabsence of hVWF for reference subtraction. After loading, all probeswere incubated in Tris buffer for 300 sec to establish a new baseline.Subsequently, biosensor probes were incubated in solutions ofFVIII-155/VWF-031, FVIIIFc Drug Substance, or rFVIII (60 nM) for 5 minat room temperature, followed by a 5 min dissociation step. Using theOctet data analysis software, the binding response (nm) was derived fromthe subtracted data (Reaction probe minus Reference probe). As shown inFIG. 15 , compared to the VWF binding affinity of rFVIIIFc and rFVIII,the VWF binding affinity of FVIII-155/VWF-031 was severely impaired.This indicates successful shielding of FVIII from full length VWF by theD′D3 fragment within the FVIIIFc/VWF heterodimer.

Example 7. VWF-FVIII Interaction is a Limiting Factor for FVIIIHalf-Life Extension

The majority of the circulating FVIII exists as a FVIII-VWF complex(>95% of plasma FVIII). This FVIII-VWF interaction promotes FVIIIclearance through the VWF clearance pathway, thus making the VWFhalf-life (T½) a limitation of the FVIII half-life extension. Toevaluate this hypothesis, the limitation of FVIII half-life extension byFc technology was tested in FVIII deficient mice (HemA mice, which haveintact VWF gene) and FVIII/VWF deficient (FVIII-VWF Double Knockout(DKO)) mice.

The HemA mice or FVIII-VWF DKO mice were treated with a singleintravenous dose of rFVIII or rFVIIIFc at 125 IU/kg in HemA mice or 200IU/kg in DKO mice. Blood samples were collected up to 72 hrs in the HemAmice or up to 8 hrs in the FVIII/VWF DKO mice. Plasma sample's FVIIIactivity was then measured by a FVIII chromogenic assay. Thepharmacokinetic (PK) profile of the two rFVIII variance was analyzedusing WinNonline program.

As shown in Table 7 and FIG. 9 , in the FVIII/VWF DKO mice, rFVIIIFcshowed about 4.8 fold longer T_(1/2) (i.e., T_(1/2) of 1.2 hr) comparedto T_(1/2) of rFVIII (i.e., T_(1/2) of 0.25 hr). In contrast, whentested in HemA mice, rFVIIIFc only had 1.8 fold longer T_(1/2) compareto rFVIII. The T_(1/2) of rFVIIIFc was 13.7 hr, which is in line withthe endogenous murine VWF half-life. This indicates that the FVIII-VWFinteraction is a limiting factor for FVIII half-life extension. In orderto achieve more than 2 fold FVIII half-life extension, the FVIII-VWFinteraction will have to be eliminated.

TABLE 7 FVIII PK in HemA and FVIIII/VWF DKO mice FVIII chromogenic assayFVIII-deficient Mice T_(1/2) Ratio FVIII/VWF-deficient Mice TestMolecule T_(1/2) (hr) vs rFVIII T_(1/2) (hr) T_(1/2) Ratio rFVIII 7.6 10.25 1 rFVIIIFc 13.7 1.8 1.2 4.8

The FVIII activity was measured using the COATEST SP FVIII kit fromDiaPharma (lot #N089019) and all incubations were performed on a 37° C.plate heater with shaking.

The range of rFVIII standard was from 100 mIU/mL to 0.78 mIU/mL. Apooled normal human plasma assay control and plasma samples (dilutedwith 1× Coatest buffer) were added into Immulon 2HB 96-well plates induplicate (25 μL/well). Freshly prepared IXa/FX/Phospholipid mix (50μL), 25 μL of 25 mM CaCl₂), and 50 μL of FXa substrate were addedsequentially into each well with 5 minutes incubation between eachaddition. After incubating with the substrate, 25 μL of 20% Acetic Acidwas added to terminate the color reaction, and the absorbance of OD405was measured with a SpectraMAX plus (Molecular Devices) instrument. Datawere analyzed with SoftMax Pro software (version 5.2). The Lowest Levelof Quantification (LLOQ) is 7.8 mIU/mL.

Example 8. VWF D′D3 Dimer Protects FVIII from FVIII Proteolysis andClearance (FIG. 10)

The FVIII protection activity of the VWF fragments was evaluated bytheir ability to protect endogenous murine FVIII from its clearance inVWF deficient mice. Different VWF fragment as listed in Table 8 Column 1(FIG. 1 , Example 1) were introduced into the blood circulation of theVWF deficient mice by Hydrodynamic injection of their corresponding DNAconstructs at 100 μg/mouse. The plasma samples were collected at 48 hrspost injection, and murine FVIII plasma activity was measured by a FVIIIchromogenic assay. VWF expression level was measured by VWF ELISA.

Four different lengths of the VWF fragments that have been tested are276, 477, 511, and 716 amino acids. The 276 to 716 amino acid range wastested to find out the length of the VWF fragments required for FVIIIbinding (276aa) without VWF's clearance receptor's binding domain(716aa). The full length VWF and the D1D2D′D3CK multimer were used asthe positive control for FVIII protection. In blood circulation, the VWFfragments synthesized with the D1D2 domain exist as a dimer and exist asmonomers when they are synthesized without the D1D2 domain.

The increase of murine FVIII activity in plasma post hydrodynamicinjection measures the FVIII protection effect of the VWF fragments. Asshown in Table 8 and FIG. 10A-B, the first 276aa of the D′D3 fragmenthad no FVIII protection activity as demonstrated by the similar pre/postinjection FVIII plasma level (FIG. 10A). However, the introduction ofthe other VWF fragments induced a significant increase on FVIII plasmalevel, indicating that those VWF fragments can protect FVIII from itsclearance pathway.

TABLE 8 FVIII/VWF DKO mice murine FVIII plasma level Pre/postintroduction of VWF fragments (DNA constructs were illustrated inFIG. 1) FVIII FVIII VWF Activity-Pre Activity-48 hr Antigen-48 hr DNAEncoding (mIU/mL) (mIU/mL) (nM/mL) CONSTRUCT VWF Fragment Avg. SD Avg.SD Avg. SD VWF-001 D′D3_(276aa) 53 31 86 16 2.8 1.9 VWF-009D2D2D′D3_(276aa) 45 20 65 17 1.8 1.3 VWF-002 D′D3_(477aa) 56 3 257 3817.0 0.5 VWF-010 D1D2D′D3_(477aa) 42 11 387 22 8.2 1.6 VWF-003D′D3A1_(511aa) 88 21 253 47 12.9 2.2 VWF-011 D1D2D′D3A1_(511aa) 63 42360 15 9.3 2.3 VWF-004 D′D3A1_(716aa) 87 8 239 56 VWF-012D1D2D′D3A1_(716aa) 64 22 307 29 VWF-006 D1D2D′D3CK 38 10 249 20 2.4 1.0VWF-008 Full length VWF 51 8 380 41 10.6 2.3

The ratio of post injection plasma FVIII activity and the plasma antigenlevel of the VWF fragments that contain the D′D3 domain of full-lengthVWF were listed in Table 8. Similar post injection FVIII/VWF ratio wasobserved from the full length VWF and the two dimer forms of the VWFfragments, meaning that those two VWF fragment dimers provide the sameFVIII protection as the full length VWF. In addition, threefold higherFVIII/VWF ratio was observed from the VWF fragment dimer isoformscompare to their corresponding monomers: the D′D3 (477aa) dimer has theFVIII/VWF ratio of 38.7 mIU/nmol; the D′D3 (477aa) monomer has theFVIII/VWF ration of 11.6 mIU/nmol: the D′D3A1 (511aa) dimer has theFVIII/VWF ratio of 32.9 mIU/nmol; and the D′D3 (511aa) monomer has theFVIII/VWF ratio of 13.8 mIU/nmol, indicating the dimer isoforms of theVWF fragments provides better FVIII protections compare to theircorresponding monomers.

TABLE 9 FVIII protection effect of full length D′D3 fragment DNAEncoding Multimer FVIII/VWF (mIU/nmol) Construct VWF Fragment State Mean(SD) VWF-002 D′D3_(477aa) Monomer 11.6 (4.4) VWF-010 D1D2D′D3_(477aa)Dimer 38.7 (11.7)  VWF-003 D′D3A1_(511aa) Monomer 13.8 (1.3) VWF-011D1D2D′D3A1_(511aa) Dimer 32.9 (5.5) VWF-008 Full length VWF Multimer31.1 (6.7)

Hydrodynamic Injection:

Hydrodynamic Injection is an efficient and safe non-viral gene deliverymethod to the liver in small animals, such as mice and rats. It wasoriginally described as a rapid injection of a naked plasmid DNA/salinesolution free of endotoxin at a tenth volume of the animal's body weightin about 5-7 seconds. The naked plasmid DNA contains the gene ofinterest and the liver produced targeted protein from the injected DNAcan be detected within 24 hours post-injection. Plasma samples were thencollected to study the therapeutic property of the expressed protein.

For all the hydrodynamic injections that were performed herein in thispatent application, 2 ml of plasmid DNA in 0.9% sterile saline solutionwas delivered via intravenous tail vein injection within about 4-7seconds to mice weighing 20-35 grams. The mice were closely monitoredfor the first couple of hours until the normal activity resumed. Afterthe blood samples were collected via retro orbital blood collection,plasma samples were then obtained and stored at −80° C. for furtheranalysis.

VWF ELISA:

Goat anti-human VWF antibody (Affinity purified, affinity biological,GAVWF-AP) was used as the capture antibody at 0.5 ug/well and VWF-EIA-D(Affinity Biologicals, VWF-EIA-D, 1:100 dilution) was used as thedetecting antibody for the VWF ELISA. ELISA assay was performedfollowing the standard ELISA procedure, TMB was used as the HRPsubstrate, PBST/1.5% BSA/0.5M NaCl buffer was used as blocking andbinding buffer. The assay standard range is 100 ng to 0.78 ng, and theassay's lowest limit of quantification (LLOQ) is 7.8 ng/mL.

Example 9: Co-Administration of Full Length VWF D′D3 Fragment ExtendrBDD-FVIII Half-Life in FVIII-VWF DKO Mice (FIG. 11)

Example 8 has demonstrated that full length D′D3 fragment can protectendogenous FVIII from its clearance pathway. In order to furtherevaluate the FVIII protection activity of D′D3 protein, FVIII-VWF DKOmice were co-administered with B domain deleted FVIII (rBDD-FVIII) andD′D3 dimer (VWF-010) or rBDD-FVIII and D′D3 monomer (VWF-002), viaintravenous injection at 200 IU/kg for rBDD-FVIII, 770 μg/kg for D′D3dimer and 590 μg/kg for D′D3 monomer. The PK profile of rBDD-FVIII wasthen monitored by its post injection plasma activity. Due to the shortin vivo half-life of the D′D3 fragments, at three hour post the initialco-injection, another dose of D′D3 was administered through the sameroute to maintain a desirable D′D3 plasma level.

For PK analysis, plasma sample was obtained via retro-orbital bloodcollection at 5 min, 30 min, 1 hour, 2 hour, 4 hour and 6 hour postinjection, plasma FVIII activity and D′D3 antigen level was analyzed byFVIII chromogenic assay and VWF ELISA.

As shown in FIG. 11 and Table 10, the D′D3 monomer prolonged rBDD-FVIIIhalf-life by 2.5 fold and improved its recovery by 1.8 fold. The D′D3dimer prolonged rBDD-FVIII half-life by 4.1 fold and improved itsrecovery by 3.5 fold. Improved mean residency time, clearance and AUCwere also observed from both of the D′D3 isoforms. The D′D3 dimer,however, achieved better results in all the PK parameters compared toits monomer form.

In summary, co-injection of full length D′D3 protects FVIII from itsclearance pathway, as show in the improved PK profile of rBDD-FVIII. Thepotential clinical value of this finding needs to be further evaluated.

TABLE 10 BDD-FVIII PK parameter in FVIII-VWF DKO mice whenco-administered with D′D3 fragments 5 min AUC_D T_(1/2) RecoveryRecovery T_(1/2) MRT Cl Vss (hr*kg*mIU/ Fold Fold Treatment (%) (hr)(hr) (mL/hr/kg) (mL/kg) mL/mIU) Increase Increase rBDD-FVIII 25 0.230.24 407.72 133.14 0.0025 rBDD-FVIII 44 0.57 0.58 151.93 124.63 0.00662.5 1.8 VWF-002 rBDD-FVIII 87 0.95 0.98 71.48 97.54 0.014 4.1 3.5VWF-010

Example 10. The D′D3 Monomer Synthesized with D1D2 Domain and its DimerIsoform have Same FVIII Protection Activity and Further ExtendedFVIIIFc's Half-Life by ˜4 Fold in FVIII-VWF DKO Mice (FIG. 12)

In order to quantify the FVIII protection ability of the D′D3 domainsand determine if the D′D3 dimerization is necessary for its FVIIIprotection activity, each of two DNA constructs (i.e., VWF-025(containing DNA sequence encoding D1D2D′D3) and VWF-029 (containingD1D2D′D3 codon DNA with C336A and C379A mutation)) was administered intoFVIII/VWF DKO mice by hydrodynamic injection. This injection resulted inD′D3 dimer (VWF-025) or monomer expression (VWF-029) in the FVIII/VWFDKO mice. At day 5 post hydrodynamic injection, a single intravenousdose of rFVIIIFc was administered at 200 IU/kg, and plasma samples wascollected at 5 min, 4, 8, 16, 24, 31, 40, 55, 66 hrs post rFVIIIFc IVinjection. An rFVIIIFc PK study that was performed in naïve FVIII-VWFDKO mice at the same dose was used as the rFVIIIFc half-life base line.Plasma FVIII activity was analyzed by a FVIII chromogenic assay. PlasmaD′D3 level was measured by VWF ELISA, and rFVIIIFc PK profile wasanalyzed using WinNonlin program.

As shown in Table 11 and FIG. 12 , with the VWF D′D3 fragments in thecirculation, rFVIIIFc's initial recovery increased from 42% to 75% withD′D3 dimer and 60% with D′D3 monomer. rFVIIIFc's T_(1/2) was alsoincreased from 2.5 hrs to 9.3 hrs and 9.2 hrs, respectively. Similar toT_(1/2), improved mean residency time, clearance, and volumedistribution were also observed from the D′D3 monomer and dimerexpressing mice. Overall, we see about 8 fold improvements on therFVIIIFc's half-life and 6 fold improvements on AUC in both D′D3 monomerand dimer expressing mice. Same as its dimer isoform, the D′D3 monomerof full-length VWF that was synthesized with propeptide (D1D2) of VWF issufficient to provide the full FVIII protection effect as the fulllength VWF molecule.

In FVIII/VWF DKO mice, WT-FVIII has a 0.25 hr T_(1/2). The Fc fusiontechnology increased FVIII T_(1/2) to 1.2 hour, which is about 4.8 foldincrease. When the Fc fusion technology was combined with the D′D3domains, the FVIII T_(1/2) was increased to 9.3 hr (D′D3 dimer) and 9.2hr (D′D3 monomer), which are about 37 fold increases in total. (Table10) This result demonstrated the synergistic effect of the Fc fusion andD′D3 VWF fragment on the FVIII half-life extension.

TABLE 11 rFVIIIFc PK parameter with/without D′D3 fragment in bloodcirculation 5 min AUC_D T_(1/2) AUC_D Recovery T_(1/2) MRT Cl Vss(hr*kg*mIU/ Fold Fold Treatment (%) (hr) (hr) (mL/hr/kg) (mL/kg) mL/mIU)Increase Increase rFVIIIFc 43 1.2 0.76 39.5 67.0 0.025 rFVIIIFc 75 9.311.1 6.1 67.6 0.164 7.8 6.6 VWF-025 rFVIIIFc 60 9.2 11.3 6.7 75.7 0.1497.7 6.0 VWF-029

Example 11: FVIII-VWF Heterodimer PK in HemA Mice

The PK profile of the lead candidates of FVIII-VWF heterodimer (such asFVIII-155/VWF-031) will be tested in HemA mice to evaluate their abilityof shielding FVIII from the endogenous VWF and their ability for FVIIIhalf-life extension.

HemA mice will be treated with a single intravenous dose of the leadcandidates at 200 IU/kg, plasma samples will then be collected at 5 min,4 hr, 8 hr, 24 hr, 48 hr, 72 hr, 96 hr and 120 hr, plasma activity willbe tested by FVIII chromogenic assay, and FVIII variance half-life willbe calculated by WinNonlin program.

In an optimal FVIII/VWF heterodimer configuration, the FVIII binding tothe endogenous VWF will be completely inhibited, therefor the base linehalf-life of rFVIII will be decreased from 7.6 hr to 0.25 hr as shown inexample 7. When D′D3 fragment non-covalently associated with FVIII,about 8 fold of half-life benefit was observed (example 9). In the leadcandidates of the FVIII/VWF heterodimer, the VWF fragment is covalentlyassociated with the FVIII molecule, better FVIII protection might beable to be achieved. The invention of this application opened the doorto further extend FVIII half-life beyond the two fold ceiling, with thecombination of the available half-life extension technologies, HemApatients could expect a better long acting FVIII variance in the nearfuture.

The PK profile of FVIII-155/VWF-031 was tested in HemA and FVIII/VWF DKOmice to evaluate the ability of the D′D3 fragment to shield the FVIIImoiety from the endogenous VWF. HemA or FVIII/VWF DKO mice were treatedwith a single intravenous dose of FVIII-155/VWF-031 at 200 IU/kg, plasmasamples were then collected at 5 min, 8 hrs, 24 hrs, and 48 hours postdosing. The FVIII activity of the plasma sample was tested by a FVIIIchromogenic assay, and the half-life of FVIII-155/VWF-031 was calculatedusing WinNonlin program.

Severely impaired binding to immobilized VWF was detected by biolayerinterferometry (FIG. 15 , Octet; ForteBio Inc., Menlo Park, Calif.) forFVIII-155/VWF-031 compared to rFVIIIFc and rFVIII. This shows the D′D3domain in the molecule had successfully blocked the FVIII binding tonative VWF molecules. Therefore, similar half-life of rFVIII-155/VWF-031was expected in the two different mouse strains. Study results arelisted in FIG. 16 and Table 12A. As predicted, rFVIII-155/VWF-031 hadcomparable PK profile in both HemA and FVIII/VWF DKO mice, indicatingthat the half-life of FVIIIFc/VWF heterodimer is independent from thehalf-life of endogenous VWF. The results show that inhibition of theinteraction between the rFVIIIFc with endogenous VWF by the VWF D′D3domains allows elimination of the FVIII half-life ceiling and opens upthe possibility of extending FVIII half-life beyond the half-lifeachievable without the VWF D′D3 domains (about two fold of the wild typeFVIII).

TABLE 12A FVIII-155/VWF-031 PK in FVIII/VWF DKO mice and HemA mice 5 minAUC_D Recovery T_(1/2) MRT Cl Vss (hr*kg*mIU/ Treatment (%) (hr) (hr)(mL/hr/kg) (mL/kg) mL/mIU) FVIII-155/ 49 9.9 6.9 11.6 80.5 0.09 VWF-031DKO FVIII-155/ 69 10.8 707 11.9 92.1 0.08 VWF-031 HemA

The FVIII protecting ability of the D′D3 domains was evaluated bycomparing the t_(1/2) of FVIII-155/VWF-031 with FVIIIFc in FVIII/VWF DKOmice. After a single IV administration, blood samples were collected at5 min, 8 hrs, 24 hrs and 48 hrs for FVIII-155/VWF-031, and at 5 min, 1hrs, 2 hrs, 4 hrs, 6 hrs and 8 hrs for FVIIIFc. The FVIII activity ofplasma sample was tested by a FVIII chromogenic assay, and the half-lifeof FVIII-155/VWF-031 was calculated using WinNonlin program.

FIG. 16B and Table 12B show a significantly improved PK profile forFVIII-155/VWF-031 compared to rFVIIIFc in DKO mice: about 6 foldincreases on t_(1/2); and about 5 fold increases in clearance and AUC.This result demonstrates that the D′D3 domain in FVIIIFc/VWF heterodimerprotects the FVIII moiety from some clearance pathways, thus providingsome of the protection normally provided by full length VWF. Thisconclusion is also confirmed in HemA mice. When compared to rFVIIIFc inHemA mice, rFVIII-155/VWF-031 has shown shorter t_(1/2) and lesser AUC,meaning in this configuration, the D′D3 domains (VWF-031) successfullyprevents binding of the FVIII protein (rFVIII-155) to endogenous VWF,which has half-life extending properties to some degree, as well as aFVIII half-life limiting property. Full length VWF is 250 kDa, and formsmultimers such that endogenous VWF can be up to 2 MDa, and therefore itis consistent with this hypothesis that the 55 kDa D′D3 region of VWFdoes not provide the same protection normally afforded by the much largeendogenous VWF in this context. Since the VWF fragment preventsendogenous VWF from binding rFVIII-155/VWF-031, in this particularconstruct the half-life is decreased in the HemA mouse. Therefore, theresults in Table 12B indicate that the rFVIII-155/VWF-031 molecule iscapable of preventing the FVIII half-life extender (endogenous VWF) frombinding the rFVIII-155/VWF-031. However, the experiment shows thatremoving the FVIII half-life limiting factor has opened up thepossibility of extending a half-life of the FVIII protein beyond 1.5fold or 2 fold shown previously. When FVIII is combined with otherhalf-life extension elements as shown in FIG. 4 , a breakthrough of the2 fold half-life extension ceiling of FVIII could be achieved.

TABLE 12B FVIII-155/VWF-031 and FVIIIFc PK in FVIII/VWF DKO mice 5 minAUC_D Recovery T_(1/2) MRT Cl Vss (hr*kg*mIU/ Treatment (%) (hr) (hr)(mL/hr/kg) (mL/kg) mL/mIU) FVIIIFc DKO 43 1.6 1.9 63.9 123.2 0.02FVIII-155/VWF- 49 9.9 6.9 11.6 80.5 0.09 031 DKO Fold Increase 6.2 3.65.5 4.5 FVIII-155/VWF- 69 10.8 7.7 11.9 92.1 0.08 031 HemA FVIIIFc HemA86 16.4 20.3 2.9 57.7 0.35

Example 12: Optimization of the D′D3-Fc Linker of FVIII/D′D3 Heterodimer(FIG. 13)

To allow rFVIIIFc to escape the VWF clearance pathway and eliminate the2 fold FVIII half-life extension ceiling, the VWF D′D3 fragment has beenincorporated into the rFVIIIFc molecule (FIG. 2 ), resulting in anFVIIIFc/VWF heterodimer. In order to eliminate the interaction betweenrFVIIIFc and endogenous VWF and maximize the D′D3 FVIII protectionpotential, the linker between the D′D3 domain and the Fc region wasadjusted to allow the optimal FVIII/D′D3 binding. A more optimal linkerwill allow the D′D3 domain to have greater FVIII protection than a lessoptimal linker construct does. This can be tested by hydrodynamicinjection of the DNA constructs in FVIII/VWF DKO mice. A more optimalconstruct will yield higher steady state protein expression of theFVIIIFc/D′D3 heterodimer.

Three different FVIIIFc/D′D3 heterodimers (FIG. 3 , Example 3) wereengineered for optimal linker selection. The possible linkers betweenthe D′D3 domains and the Fc region were listed in Table 13. Those DNAconstructs were administered into FVIII/VWF DKO mice by hydrodynamicinjection (“HDI”) at 100 μg/mouse, and plasma samples were collected 48hr post HDI. Circulating FVIIIFc/D′D3 heterodimer activity was analyzedby a FVIII chromogenic assay.

The study result was shown in FIG. 13 . 48 hours post HDI, similarexpression level were reached by FVIII-064 and FVIII-159, indicating the20aa linker and the 35aa linker promote similar level of FVIII/D′D3interaction. In another hand, FVIII-160 showed significantly higherexpression than FVIII-064, meaning that the 48aa linker allows betterFVIII/D′D3 binding compare to the 20aa and 35aa linkers.

An optimal linker between the VWF fragment and the Fc region is one ofthe key elements of the FVIIIFc/VWF heterodimer. Finding the best linkerwill allow the optimal interaction between FVIII and the VWF fragment,prevent FVIII binding to endogenous VWF, enable FVIII to escape the VWFclearance pathway, and extend the FVIII half-life beyond the plasma VWFhalf-life.

TABLE 13 Different linkers between D′D3 and Fc fragment DNA constructLinker between D′D3 and Fc FVIII-064 20 aa = I D G G G G S G G G(SEQ ID NO: G S L V P R G S G G 92) FVIII-159 35 aa = (SEQ ID NO:I S G G G G S G G G G S G G 93) G G S G G G G S G G G G S LV P R G S G G FVIII-160 48 aa = (SEQ ID NO: I S G G G G S G G G G S G G94) G G S G G G G S G G G G S G G G G S L V P R G S G G G G S G G G G S

Example 13: Single Chain FVIII Stability

The Single chain FVIII protein might be more stable than its dual chainisoform. To test this hypothesis, two DNA constructs were made:FVIII-136 (processable FVIIIFc with the D′D3 domain) and FVIII-148(Single Chain (SC) FVIIIFc with the D′D3 domain, which containsR1645A/R1648A mutation to prevent cleavage between FVIII heavy chain andlight chain).

Both plasmids were administered into FVIII/VWF DKO mice by hydrodynamicinjection. Plasma samples were collected 24 hr and 48 hr post injectionsto measure the expression level of the two FVIIIFc/D′D3 isoforms. Asshown in FIG. 14 , at both time points, a trend of better expression wasobserved by the SC-FVIIIFc/D′D3 construct (FVIII-148) (p=0.12, p=0.19),indicating single chain FVIII might be more stable or better expressedthan its dual chain isoform (FVIII-136). The PK profile of the two FVIIIisoforms and their cell culture expression levels will be furtherinvestigated. The single chain FVIII isoform could be potentially usedto replace the conventional dual chain isoform to achieve better proteinproduction and better in vivo FVIII half-life.

Example 14. PEGylation

One or more polyethylene glycol (PEG) molecules can be attached withinany regions of the FVIII protein, the VWF fragment, or both. As FVIIIdoes not have a free cysteine at its surface based on crystal structure(PDB:2R7E, Shen et al., Blood 111:1240 (2008); PDB:3CDZ, Ngo, Structure,16:597-606 (2008)), one approach is to insert a cysteine containingpeptide (e.g., GGGSGCGGGS) (SEQ ID NO: 107) into or link it to the FVIIIprotein, the VWF fragment, or both. PEG molecules containing maleimidecan then be conjugated specifically to the cysteine introduced on therecombinant FVIII protein. Briefly, the recombinant FVIII proteincontaining the Cys insertion can be constructed by standard moleculartechnology, and the recombinant FVIII protein expressed in mammalianexpression system (e.g., HEK293, CHO, BHK21, PER.C6, and CAP cells) canbe purified via affinity and ion exchange chromatography. The purifiedrecombinant FVIII protein is reduced by Tris(2-carboxyethyl)phosphine(TCEP) to expose the thiol group of the introduced cysteine and thenreacted with maleimide PEG. The resulting recombinant FVIII protein istested for procoagulant activity and extended half-life.

PEG is attached to at least one of the locations disclosed in U.S. Appl.No. 61/670,553, which is incorporated herein by reference in itsentirety, or other suitable insertion sites. The FVIII activity of thePEGylated recombinant FVIII protein is analyzed using a FVIIIchromogenic assay. The PK of the PEGylated recombinant FVIII protein isanalyzed in HemA mice and FVIII-VWF DKO Mice as described above.

Example 15: FVIII Stability in HemA and FVIII/VWF Double Knockout (DKO)Plasma

Plasma stability of different FVIIIFc fusions was tested in HemA orFVIII/VWF double knockout (DKO) plasma. For stability assay, 5 IU/ml ofvarious FVIIIFc proteins were incubated with either mouse HemA or DKOplasma at 37° C. Aliquots were collected at different time points tomeasure activity by FVIII chromogenic assay. Activity at each time pointwas measured in duplicate and average activity was plotted as a functionof time.

For the FVIIIFc immuno-precipitation assay, 5 μg FVIIIFc was incubatedwith either 250 μl of PBS or mouse DKO plasma for 24 hrs at 37° C.FVIIIFc was immuno-precipitated by adding 5 μg sheep anti-FVIIIpolyclonal antibody (ab61370) for 1 hr at room temperature and 100 μlprotein A beads. After 4×1 ml PBS washes, beads were re-suspended in 50μl 1× reducing SDS-PAGE buffer. After boiling, 20 μl sample (i.e. ˜1 gFVIIIFc) was loaded on to 4-15% Bio-Rad stain free gel. Gel was imagedby Bio-rad system followed by western analysis with FVIII anti heavychain antibody (GMA012).

Activity of FVIIIFc (dual chain FVIII molecule, which has separate FVIIIheavy and light chains, held together by non-covalent interactions)decreases with time in both HemA and DKO plasma (FIG. 18A). Due to lackof VWF mediated protection, loss in FVIIIFc activity was more pronouncedin DKO plasma. This loss in FVIII activity was mainly due todissociation or degradation of FVIII heavy chain (HC). About a 75%reduction in FVIIIFc heavy chain was observed after a 24 hr incubationin DKO plasma (FIG. 18B). No significant reduction was observed foreither light chain (LC) (data not shown) or non-processed/single chainFVIIIFc (i.e. FVIII molecule in which light chain and heavy chain arestill held together covalently-top band in the gel picture) (FIG. 18B).

As VWF is proposed to increase the stability of FVIII in vivo, we testedif chimeric protein-FVIII-VWF heterodimer (FVIII155:VWF31, which has VWFD′D3 covalently, attached to FVIII through Fc) was more stable in Hem Aand DKO plasma. From plasma stability data shown in FIG. 19 , thepresence of D′D3 increased the stability of FVIIIFc, both in HemA andDKO plasma. Single chain FVIIIFc without D′D3 was used as control inthese experiments (scFVIII). From FIG. 19 , single chain FVIII was morestable than dual chain FVIIIFc; however the presence of D′D3significantly increased the plasma stability of single chain FVIIIFcmolecule further. This suggests that D′D3 stabilizes FVIII, not just byholding heavy and light chain together but also through some otherunknown mechanisms.

Example 16: Use of Furin/PACE for VWF Processing

VWF is a unique protein in the sense that it contains a very largepro-peptide (i.e. D1D2 domain of VWF, ˜85 kDa). The VWF pro-peptideserves as an internal chaperone for proper folding of VWF molecule. Twoenzymes were tested for VWF processing-PC5 and Furin (PACE). VWF031construct (D1D2D′D3Fc) was transiently co-transfected in HEK293 cellswith various concentrations of either PC5 or PACE. After four days, thetissue culture media was collected and subjected to protein A pull down.Even at a lower concentration (2.5%), furin (PACE) was more efficientthan 10% PC5, in removing the pro-peptide (D1D2) from D′D3Fc (FIG. 20 ).Removal of D1D2 is important, as the presence of D1D2 has beenimplicated in preventing interaction of D′D3 with FVIII.

Example 17: VWF Fragment in FVIII-VWF Heterodimer Prevents FVIIIInteraction with Full Length VWF

A ForteBio octet instrument was used to test FVIII construct 155/VWF31heterodimer binding to full length VWF (FIG. 21A). For the bindingassay, full length VWF was captured by using APS sensor, followed byblocking with 1% BSA. After blocking, different FVIII constructs weretested for VWF binding. As predicted, wild type FVIII and FVIIIFc boundstrongly to the VWF sensors. FVIII Y1680F mutant, which is known to havelow or no affinity for VWF showed significantly reduced VWF binding.FVIII155/VWF31 heterodimer did not bind at all to full length VWF,confirming shielding of FVIII with D′D3 in FVIII-VWF heterodimer.

The same experiment was performed in reverse orientation to determine ifthe D′D3 portion in the FVIII-VWF heterodimer can interact with otherFVIII molecules not covalently attached to D′D3. As shown in FIG. 21B,the VWF31 (D′D3Fc) construct alone when immobilized on protein G sensorcan bind strongly to FVIII, however the D′D3 in FVIII155:VWF31heterodimer did not show any binding to FVIII. Protein G alone withFVIII was used as control. These binding experiments confirmed that D′D3in heterodimer can interact with only one FVIII molecule which iscovalently attached to it and prevent FVIII from interacting with fulllength wild type VWF molecules.

To determine the exact binding affinity of VWF D′D3 for FVIII molecule,surface plasma resonance experiments were performed with VWF031 (FIG. 22). VWF031 construct (D′D3Fc) was captured by using anti-human IgG andB-domain deleted FVIII was passed over D′D3Fc containing chip. A K_(D)of about 10 nM was observed for FVIII. This affinity is about 25-foldlower compare to full length wild type VWF molecule and is similar towhat is reported previously in literature.

Example 18: Effect of Different Linker Length in Between D′D3 and Fc onHeterodimer Activity and PK

To check if varying the length of thrombin cleavable linker in betweenD′D3 and Fc has any effect on the PK and activity of FVIII-VWFheterodimer, different VWF constructs were co-expressed along with FVIII155. Three different linker lengths constructs listed in Table 14A weretested (VWF031, VWF035 and VWF036). Each plasmid was mixed with FVIII155plasmid (Example 5) and transfected into HEK293 cells. At day four posttransfection, cell culture media was harvested and concentrated to 10IU/ml FVIII chromogenic activity.

Concentrated cell media was then administered into 8-12 weeks oldFVIII/VWF DKO mice at 100 IU/10 mL/kg dose. Plasma samples werecollected at 5 min, 8 hr, 16 hr, 24 hr, 32 hr and 48 hr post dosing.FVIII activity of the plasma samples were analyzed by FVIII chromogenicassay and half-life was calculated using WinNonlin-Phoenix program.

As shown in FIG. 23 , when the linker length between D′D3 and Fcfragment was increased from 48 aa to 73aa or 98aa, the half-life of thecorresponding FVIIIc/VWF heterodimer increased and reached 12.2 hr and13.3 hr respectively. This represents a 1.5 to 1.6 fold increase over48aa long variant. To date, the 98aa linker is the most optimal linkerto utilize the FVIII protection activity of the D′D3 fragment, and itwill be incorporated into FVIIIFc/VWF heterodimer to further improve itshalf-life.

To compare the effect of linker on FVIII activity, FVIII chromogenic andaPTT assay were performed on tissue culture media from cells expressingdifferent FVIII-VWF heterodimers. Though aPTT activity was 2-foldreduced compare to chromogenic activity for heterodimer constructs, nosignificant difference was seen between various linkers, except when thelinker also contain a PAR1 site next to thrombin site (Table 14B).

TABLE 14A Sequence of Variable Linker in between VWF D′D3 and FcDNA construct Linker between D′D3 and Fc VWF031 48 aa =I S G G G G S G G G G S G G G G S G G G G S G G G G S G G G G SL V P R G S G G G G S G G G G S (SEQ ID NO: 95) VWF035 73aa =I S G G G G S G G G G S G G G G S G G G G S G G G G S G G G G SG G G G S G G G G S G G G G S G G G G S G G G G S L V P R G S GG G G S G G G G S (SEQ ID NO: 96) VWF036 98aa =I S G G G G S G G G G S G G G G S G G G G S G G G G S G G G G SG G G G S G G G G S G G G G S G G G G S G G G G S G G G G S G GG G S G G G G S G G G G S G G G G S L V P R G S G G G G S G G GG S  (SEQ ID NO: 97)

TABLE 14B Heterodimer activity with different linker length Linkerlength Sample between D′D3 Chromogenic aPTT Chromogenic/ ID Sampledescription and Fc (aa) IU/mL IU/mL aPTT 1 FVIII Fc 155 + VWF15 20 1.810.85 2.14 2 FVIII Fc 155 + VWF31 48 2.32 1.05 2.21 3 FVIII Fc 155 +VWF33 35 2.21 1.02 2.16 4 FVIII Fc 155 + VWF35 73 2.65 1.24 2.14 5 FVIIIFc 155 + VWF36 98 2.75 1.11 2.47 6 FVIII Fc 155 + VWF39 26 1.85 1.211.53 (thrombin + PAR1)

Example 19: Linking FVIII with VWF Fragment Using Sortase Enzyme

In another aspect, a VWF fragment (e.g. D1D2D′D3 or D′D3 domain) isattached to FVIII by using sortase mediated in vitro protein ligationmethod. In one example, Staphylococcus aureus sortase A (LPXTG)recognition motif was introduced at the C-terminus of VWF fragment andGly(n) residue at the N-terminus of FVIII (where the number of glycineresidues is variable). The FVIII molecule used can be either singlechain or dual chain. The sortase catalyzed trans-peptidation reactionwill covalently attach the VWF fragment to FVIII. Reverse orientation ofrecognition motif can also be used to link these two proteins, where wehave FVIII at the N-terminus with LPXTG motif and VWF fragment at theC-terminus with Gly(n) (See FIG. 24 —example of sortase ligation forreference). The LPXTG motif and Glycine residues can be replaced withother sortase recognition sequences.

VWF fragment containing sortase A recognition sequence Fc fusion proteinwas also made. For Fc fusion constructs, VWF D1D2D′D3 fragment was fusedwith Fc region of IgG through a GS linker that contains a sortaserecognition sequence and a thrombin cleavage site (Table 15 and 16).Once protein is expressed and purified on Protein A column, the Fcregion can be removed by thrombin cleavage. Resulting VWF fragment withsortase A recognition site can then be used for ligation with FVIIImolecule (FIG. 24 —Example of sortase ligation for reference—row E).

pSYN-VWF-051 has a 54 amino acids linker with sortase and thrombin sitein between the VWF fragment and the Fc region. Synthesis of DNA fragmentcoding for 54 amino acids linker (ISGGGGSGGG GSGGGGSGGG GSGGGGSGGGGSLPETGALR PRVVGGGGSG GGGS) (SEQ ID NO: 98) and a portion of the Fcregion was outsourced (Genewiz Sequence no-10-210746313, shown below). Afragment of the Genewiz construct was sub cloned into the EcoRV/RsRIIdigested pSYN-VWF-031.

Gene wiz-Sequence no-10-210746313 (SEQ ID NO: 99)AGGAGCCGATATCTGGCGGTGGAGGTTCCGGTGGCGGGGGATCCGGCGGTGGAGGTTCCGGCGGTGGAGGTTCCGGTGGCGGGGGATCCGGTGGCGGGGGATCCTTACCTGAAACTGGAGCCCTGCGGCCCCGGGTCGTCGGCGGTGGAGGTTCCGGTGGCGGGGGATCCGACAAAACTCACACATGCCCACCGTGCCCAGCTCCAGAACTCCTGG GCGGACCGTCAGTCTT

The sequence of N-terminus pentaglycine containing single chain FVIII isshown in Table 17 and 18.

TABLE 15Nucleotide sequence of pSYN-VWF051 (VWF DlD2D′D3Fc with sortase Arecognition motif and thrombin cleavable linker in between VWFfragment and Fc) (SEQ ID NO: 100)   1 ATGATTCCTG CCAGATTTGC CGGGGTGCTG CTTGCTCTGG CCCTCATTTT  51 GCCAGGGACC CTTTGTGCAG AAGGAACTCG CGGCAGGTCA TCCACGGCCC 101 GATGCAGCCT TTTCGGAAGT GACTTCGTCA ACACCTTTGA TGGGAGCATG 151 TACAGCTTTG CGGGATACTG CAGTTACCTC CTGGCAGGGG GCTGCCAGAA 201 ACGCTCCTTC TCGATTATTG GGGACTTCCA GAATGGCAAG AGAGTGAGCC 251 TCTCCGTGTA TCTTGGGGAA TTTTTTGACA TCCATTTGTT TGTCAATGGT 301 ACCGTGACAC AGGGGGACCA AAGAGTCTCC ATGCCCTATG CCTCCAAAGG 351 GCTGTATCTA GAAACTGAGG CTGGGTACTA CAAGCTGTCC GGTGAGGCCT 401 ATGGCTTTGT GGCCAGGATC GATGGCAGCG GCAACTTTCA AGTCCTGCTG 451 TCAGACAGAT ACTTCAACAA GACCTGCGGG CTGTGTGGCA ACTTTAACAT 501 CTTTGCTGAA GATGACTTTA TGACCCAAGA AGGGACCTTG ACCTCGGACC 551 CTTATGACTT TGCCAACTCA TGGGCTCTGA GCAGTGGAGA ACAGTGGTGT 601 GAACGGGCAT CTCCTCCCAG CAGCTCATGC AACATCTCCT CTGGGGAAAT 651 GCAGAAGGGC CTGTGGGAGC AGTGCCAGCT TCTGAAGAGC ACCTCGGTGT 701 TTGCCCGCTG CCACCCTCTG GTGGACCCCG AGCCTTTTGT GGCCCTGTGT 751 GAGAAGACTT TGTGTGAGTG TGCTGGGGGG CTGGAGTGCG CCTGCCCTGC 801 CCTCCTGGAG TACGCCCGGA CCTGTGCCCA GGAGGGAATG GTGCTGTACG 851 GCTGGACCGA CCACAGCGCG TGCAGCCCAG TGTGCCCTGC TGGTATGGAG 901 TATAGGCAGT GTGTGTCCCC TTGCGCCAGG ACCTGCCAGA GCCTGCACAT 951 CAATGAAATG TGTCAGGAGC GATGCGTGGA TGGCTGCAGC TGCCCTGAGG1001 GACAGCTCCT GGATGAAGGC CTCTGCGTGG AGAGCACCGA GTGTCCCTGC1051 GTGCATTCCG GAAAGCGCTA CCCTCCCGGC ACCTCCCTCT CTCGAGACTG1101 CAACACCTGC ATTTGCCGAA ACAGCCAGTG GATCTGCAGC AATGAAGAAT1151 GTCCAGGGGA GTGCCTTGTC ACTGGTCAAT CCCACTTCAA GAGCTTTGAC1201 AACAGATACT TCACCTTCAG TGGGATCTGC CAGTACCTGC TGGCCCGGGA1251 TTGCCAGGAC CACTCCTTCT CCATTGTCAT TGAGACTGTC CAGTGTGCTG1301 ATGACCGCGA CGCTGTGTGC ACCCGCTCCG TCACCGTCCG GCTGCCTGGC1351 CTGCACAACA GCCTTGTGAA ACTGAAGCAT GGGGCAGGAG TTGCCATGGA1401 TGGCCAGGAC ATCCAGCTCC CCCTCCTGAA AGGTGACCTC CGCATCCAGC1451 ATACAGTGAC GGCCTCCGTG CGCCTCAGCT ACGGGGAGGA CCTGCAGATG1501 GACTGGGATG GCCGCGGGAG GCTGCTGGTG AAGCTGTCCC CCGTCTATGC1551 CGGGAAGACC TGCGGCCTGT GTGGGAATTA CAATGGCAAC CAGGGCGACG1601 ACTTCCTTAC CCCCTCTGGG CTGGCGGAGC CCCGGGTGGA GGACTTCGGG1651 AACGCCTGGA AGCTGCACGG GGACTGCCAG GACCTGCAGA AGCAGCACAG1701 CGATCCCTGC GCCCTCAACC CGCGCATGAC CAGGTTCTCC GAGGAGGCGT1751 GCGCGGTCCT GACGTCCCCC ACATTCGAGG CCTGCCATCG TGCCGTCAGC1801 CCGCTGCCCT ACCTGCGGAA CTGCCGCTAC GACGTGTGCT CCTGCTCGGA1851 CGGCCGCGAG TGCCTGTGCG GCGCCCTGGC CAGCTATGCC GCGGCCTGCG1901 CGGGGAGAGG CGTGCGCGTC GCGTGGCGCG AGCCAGGCCG CTGTGAGCTG1951 AACTGCCCGA AAGGCCAGGT GTACCTGCAG TGCGGGACCC CCTGCAACCT2001 GACCTGCCGC TCTCTCTCTT ACCCGGATGA GGAATGCAAT GAGGCCTGCC2051 TGGAGGGCTG CTTCTGCCCC CCAGGGCTCT ACATGGATGA GAGGGGGGAC2101 TGCGTGCCCA AGGCCCAGTG CCCCTGTTAC TATGACGGTG AGATCTTCCA2151 GCCAGAAGAC ATCTTCTCAG ACCATCACAC CATGTGCTAC TGTGAGGATG2201 GCTTCATGCA CTGTACCATG AGTGGAGTCC CCGGAAGCTT GCTGCCTGAC2251 GCTGTCCTCA GCAGTCCCCT GTCTCATCGC AGCAAAAGGA GCCTATCCTG2301 TCGGCCCCCC ATGGTCAAGC TGGTGTGTCC CGCTGACAAC CTGCGGGCTG2351 AAGGGCTCGA GTGTACCAAA ACGTGCCAGA ACTATGACCT GGAGTGCATG2401 AGCATGGGCT GTGTCTCTGG CTGCCTCTGC CCCCCGGGCA TGGTCCGGCA2451 TGAGAACAGA TGTGTGGCCC TGGAAAGGTG TCCCTGCTTC CATCAGGGCA2501 AGGAGTATGC CCCTGGAGAA ACAGTGAAGA TTGGCTGCAA CACTTGTGTC2551 TGTCGGGACC GGAAGTGGAA CTGCACAGAC CATGTGTGTG ATGCCACGTG2601 CTCCACGATC GGCATGGCCC ACTACCTCAC CTTCGACGGG CTCAAATACC2651 TGTTCCCCGG GGAGTGCCAG TACGTTCTGG TGCAGGATTA CTGCGGCAGT2701 AACCCTGGGA CCTTTCGGAT CCTAGTGGGG AATAAGGGAT GCAGCCACCC2751 CTCAGTGAAA TGCAAGAAAC GGGTCACCAT CCTGGTGGAG GGAGGAGAGA2801 TTGAGCTGTT TGACGGGGAG GTGAATGTGA AGAGGCCCAT GAAGGATGAG2851 ACTCACTTTG AGGTGGTGGA GTCTGGCCGG TACATCATTC TGCTGCTGGG2901 CAAAGCCCTC TCCGTGGTCT GGGACCGCCA CCTGAGCATC TCCGTGGTCC2951 TGAAGCAGAC ATACCAGGAG AAAGTGTGTG GCCTGTGTGG GAATTTTGAT3001 GGCATCCAGA ACAATGACCT CACCAGCAGC AACCTCCAAG TGGAGGAAGA3051 CCCTGTGGAC TTTGGGAACT CCTGGAAAGT GAGCTCGCAG TGTGCTGACA3101 CCAGAAAAGT GCCTCTGGAC TCATCCCCTG CCACCTGCCA TAACAACATC3151 ATGAAGCAGA CGATGGTGGA TTCCTCCTGT AGAATCCTTA CCAGTGACGT3201 CTTCCAGGAC TGCAACAAGC TGGTGGACCC CGAGCCATAT CTGGATGTCT3251 GCATTTACGA CACCTGCTCC TGTGAGTCCA TTGGGGACTG CGCCGCATTC3301 TGCGACACCA TTGCTGCCTA TGCCCACGTG TGTGCCCAGC ATGGCAAGGT3351 GGTGACCTGG AGGACGGCCA CATTGTGCCC CCAGAGCTGC GAGGAGAGGA3401 ATCTCCGGGA GAACGGGTAT GAGGCTGAGT GGCGCTATAA CAGCTGTGCA3451 CCTGCCTGTC AAGTCACGTG TCAGCACCCT GAGCCACTGG CCTGCCCTGT3501 GCAGTGTGTG GAGGGCTGCC ATGCCCACTG CCCTCCAGGG AAAATCCTGG3551 ATGAGCTTTT GCAGACCTGC GTTGACCCTG AAGACTGTCC AGTGTGTGAG3601 GTGGCTGGCC GGCGTTTTGC CTCAGGAAAG AAAGTCACCT TGAATCCCAG3651 TGACCCTGAG CACTGCCAGA TTTGCCACTG TGATGTTGTC AACCTCACCT3701 GTGAAGCCTG CCAGGAGCCG ATATCTGGCG GTGGAGGTTC CGGTGGCGGG3751 GGATCCGGCG GTGGAGGTTC CGGCGGTGGA GGTTCCGGTG GCGGGGGATC3801 CGGTGGCGGG GGATCCTTAC CTGAAACTGG AGCCCTGCGG CCCCGGGTCG3851 TCGGCGGTGG AGGTTCCGGT GGCGGGGGAT CCGACAAAAC TCACACATGC3901 CCACCGTGCC CAGCTCCAGA ACTCCTGGGC GGACCGTCAG TCTTCCTCTT3951 CCCCCCAAAA CCCAAGGACA CCCTCATGAT CTCCCGGACC CCTGAGGTCA4001 CATGCGTGGT GGTGGACGTG AGCCACGAAG ACCCTGAGGT CAAGTTCAAC4051 TGGTACGTGG ACGGCGTGGA GGTGCATAAT GCCAAGACAA AGCCGCGGGA4101 GGAGCAGTAC AACAGCACGT ACCGTGTGGT CAGCGTCCTC ACCGTCCTGC4151 ACCAGGACTG GCTGAATGGC AAGGAGTACA AGTGCAAGGT CTCCAACAAA4201 GCCCTCCCAG CCCCCATCGA GAAAACCATC TCCAAAGCCA AAGGGCAGCC4251 CCGAGAACCA CAGGTGTACA CCCTGCCCCC ATCCCGGGAT GAGCTGACCA4301 AGAACCAGGT CAGCCTGACC TGCCTGGTCA AAGGCTTCTA TCCCAGCGAC4351 ATCGCCGTGG AGTGGGAGAG CAATGGGCAG CCGGAGAACA ACTACAAGAC4401 CACGCCTCCC GTGTTGGACT CCGACGGCTC CTTCTTCCTC TACAGCAAGC4451 TCACCGTGGA CAAGAGCAGG TGGCAGCAGG GGAACGTCTT CTCATGCTCC4501 GTGATGCATG AGGCTCTGCA CAACCACTAC ACGCAGAAGA GCCTCTCCCT4551 GTCTCCGGGT AAATGA

TABLE 16 Protein sequence of VWF051 (VWF DlD2D′D3Fcwith sortase A recognition motif and thrombincleavable linker in between VWF fragmentand Fc; sortase A site shown in bold) (SEQ ID NO: 101)   1 MIPARFAGVL LALALILPGT LCAEGTRGRS STARCSLFGS DFVNTFDGSM  51 YSFAGYCSYL LAGGCQKRSF SIIGDFQNGK RVSLSVYLGE FFDIHLFVNG 101 TVTQGDQRVS MPYASKGLYL ETEAGYYKLS GEAYGFVARI DGSGNFQVLL 151 SDRYFNKTCG LCGNFNIFAE DDFMTQEGTL TSDPYDFANS WALSSGEQWC 201 ERASPPSSSC NISSGEMQKG LWEQCQLLKS TSVFARCHPL VDPEPFVALC 251 EKTLCECAGG LECACPALLE YARTCAQEGM VLYGWTDHSA CSPVCPAGME 301 YRQCVSPCAR TCQSLHINEM CQERCVDGCS CPEGQLLDEG LCVESTECPC 351 VHSGKRYPPG TSLSRDCNTC ICRNSQWICS NEECPGECLV TGQSHFKSFD 401 NRYFTFSGIC QYLLARDCQD HSFSIVIETV QCADDRDAVC TRSVTVRLPG 451 LHNSLVKLKH GAGVAMDGQD IQLPLLKGDL RIQHTVTASV RLSYGEDLQM 501 DWDGRGRLLV KLSPVYAGKT CGLCGNYNGN QGDDFLTPSG LAEPRVEDFG 551 NAWKLHGDCQ DLQKQHSDPC ALNPRMTRFS EEACAVLTSP TFEACHRAVS 601 PLPYLRNCRY DVCSCSDGRE CLCGALASYA AACAGRGVRV AWREPGRCEL 651 NCPKGQVYLQ CGTPCNLTCR SLSYPDEECN EACLEGCFCP PGLYMDERGD 701 CVPKAQCPCY YDGEIFQPED IFSDHHTMCY CEDGFMHCTM SGVPGSLLPD 751 AVLSSPLSHR SKRSLSCRPP MVKLVCPADN LRAEGLECTK TCQNYDLECM 801 SMGCVSGCLC PPGMVRHENR CVALERCPCF HQGKEYAPGE TVKIGCNTCV 851 CRDRKWNCTD HVCDATCSTI GMAHYLTFDG LKYLFPGECQ YVLVQDYCGS 901 NPGTFRILVG NKGCSHPSVK CKKRVTILVE GGEIELFDGE VNVKRPMKDE 951 THFEVVESGR YIILLLGKAL SVVWDRHLSI SVVLKQTYQE KVCGLCGNFD1001 GIQNNDLTSS NLQVEEDPVD FGNSWKVSSQ CADTRKVPLD SSPATCHNNI1051 MKQTMVDSSC RILTSDVFQD CNKLVDPEPY LDVCIYDTCS CESIGDCAAF1101 CDTIAAYAHV CAQHGKVVTW RTATLCPQSC EERNLRENGY EAEWRYNSCA1151 PACQVTCQHP EPLACPVQCV EGCHAHCPPG KILDELLQTC VDPEDCPVCE1201 VAGRRFASGK KVTLNPSDPE HCQICHCDVV NLTCEACQEP ISGGGGSGGG1251 GSGGGGSGGG GSGGGGSGGG GSLPETGALR PRVVGGGGSG GGGSDKTHTC1301 PPCPAPELLG GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVKFN1351 WYVDGVEVHN AKTKPREEQY NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK1401 ALPAPIEKTI SKAKGQPREP QVYTLPPSRD ELTKNQVSLT CLVKGFYPSD1451 IAVEWESNGQ PENNYKTTPP VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS1501 VMHEALHNHY TQKSLSLSPG K*

TABLE 17 Nucleotide sequence of FVIII 265 (FVIII singlechain molecule with pentaglycines at N-terminus) (SEQ ID NO: 102)   1 ATGCAAATAG AGCTCTCCAC CTGCTTCTTT CTGTGCCTTT TGCGATTCTG  51 CTTTAGTGGA GGAGGAGGAG GAGCCACCAG AAGATACTAC CTGGGTGCAG 101 TGGAACTGTC ATGGGACTAT ATGCAAAGTG ATCTCGGTGA GCTGCCTGTG 151 GACGCAAGAT TTCCTCCTAG AGTGCCAAAA TCTTTTCCAT TCAACACCTC 201 AGTCGTGTAC AAAAAGACTC TGTTTGTAGA ATTCACGGAT CACCTTTTCA 251 ACATCGCTAA GCCAAGGCCA CCCTGGATGG GTCTGCTAGG TCCTACCATC 301 CAGGCTGAGG TTTATGATAC AGTGGTCATT ACACTTAAGA ACATGGCTTC 351 CCATCCTGTC AGTCTTCATG CTGTTGGTGT ATCCTACTGG AAAGCTTCTG 401 AGGGAGCTGA ATATGATGAT CAGACCAGTC AAAGGGAGAA AGAAGATGAT 451 AAAGTCTTCC CTGGTGGAAG CCATACATAT GTCTGGCAGG TCCTGAAAGA 501 GAATGGTCCA ATGGCCTCTG ACCCACTGTG CCTTACCTAC TCATATCTTT 551 CTCATGTGGA CCTGGTAAAA GACTTGAATT CAGGCCTCAT TGGAGCCCTA 601 CTAGTATGTA GAGAAGGGAG TCTGGCCAAG GAAAAGACAC AGACCTTGCA 651 CAAATTTATA CTACTTTTTG CTGTATTTGA TGAAGGGAAA AGTTGGCACT 701 CAGAAACAAA GAACTCCTTG ATGCAGGATA GGGATGCTGC ATCTGCTCGG 751 GCCTGGCCTA AAATGCACAC AGTCAATGGT TATGTAAACA GGTCTCTGCC 801 AGGTCTGATT GGATGCCACA GGAAATCAGT CTATTGGCAT GTGATTGGAA 851 TGGGCACCAC TCCTGAAGTG CACTCAATAT TCCTCGAAGG TCACACATTT 901 CTTGTGAGGA ACCATCGCCA GGCGTCCTTG GAAATCTCGC CAATAACTTT 951 CCTTACTGCT CAAACACTCT TGATGGACCT TGGACAGTTT CTACTGTTTT1001 GTCATATCTC TTCCCACCAA CATGATGGCA TGGAAGCTTA TGTCAAAGTA1051 GACAGCTGTC CAGAGGAACC CCAACTACGA ATGAAAAATA ATGAAGAAGC1101 GGAAGACTAT GATGATGATC TTACTGATTC TGAAATGGAT GTGGTCAGGT1151 TTGATGATGA CAACTCTCCT TCCTTTATCC AAATTCGCTC AGTTGCCAAG1201 AAGCATCCTA AAACTTGGGT ACATTACATT GCTGCTGAAG AGGAGGACTG1251 GGACTATGCT CCCTTAGTCC TCGCCCCCGA TGACAGAAGT TATAAAAGTC1301 AATATTTGAA CAATGGCCCT CAGCGGATTG GTAGGAAGTA CAAAAAAGTC1351 CGATTTATGG CATACACAGA TGAAACCTTT AAGACTCGTG AAGCTATTCA1401 GCATGAATCA GGAATCTTGG GACCTTTACT TTATGGGGAA GTTGGAGACA1451 CACTGTTGAT TATATTTAAG AATCAAGCAA GCAGACCATA TAACATCTAC1501 CCTCACGGAA TCACTGATGT CCGTCCTTTG TATTCAAGGA GATTACCAAA1551 AGGTGTAAAA CATTTGAAGG ATTTTCCAAT TCTGCCAGGA GAAATATTCA1601 AATATAAATG GACAGTGACT GTAGAAGATG GGCCAACTAA ATCAGATCCT1651 CGGTGCCTGA CCCGCTATTA CTCTAGTTTC GTTAATATGG AGAGAGATCT1701 AGCTTCAGGA CTCATTGGCC CTCTCCTCAT CTGCTACAAA GAATCTGTAG1751 ATCAAAGAGG AAACCAGATA ATGTCAGACA AGAGGAATGT CATCCTGTTT1801 TCTGTATTTG ATGAGAACCG AAGCTGGTAC CTCACAGAGA ATATACAACG1851 CTTTCTCCCC AATCCAGCTG GAGTGCAGCT TGAGGATCCA GAGTTCCAAG1901 CCTCCAACAT CATGCACAGC ATCAATGGCT ATGTTTTTGA TAGTTTGCAG1951 TTGTCAGTTT GTTTGCATGA GGTGGCATAC TGGTACATTC TAAGCATTGG2001 AGCACAGACT GACTTCCTTT CTGTCTTCTT CTCTGGATAT ACCTTCAAAC2051 ACAAAATGGT CTATGAAGAC ACACTCACCC TATTCCCATT CTCAGGAGAA2101 ACTGTCTTCA TGTCGATGGA AAACCCAGGT CTATGGATTC TGGGGTGCCA2151 CAACTCAGAC TTTCGGAACA GAGGCATGAC CGCCTTACTG AAGGTTTCTA2201 GTTGTGACAA GAACACTGGT GATTATTACG AGGACAGTTA TGAAGATATT2251 TCAGCATACT TGCTGAGTAA AAACAATGCC ATTGAACCAA GAAGCTTCTC2301 TCAAAACCCA CCAGTCTTGA AGGCCCATCA GGCCGAAATA ACTCGTACTA2351 CTCTTCAGTC AGATCAAGAG GAAATTGACT ATGATGATAC CATATCAGTT2401 GAAATGAAGA AGGAAGATTT TGACATTTAT GATGAGGATG AAAATCAGAG2451 CCCCCGCAGC TTTCAAAAGA AAACACGACA CTATTTTATT GCTGCAGTGG2501 AGAGGCTCTG GGATTATGGG ATGAGTAGCT CCCCACATGT TCTAAGAAAC2551 AGGGCTCAGA GTGGCAGTGT CCCTCAGTTC AAGAAAGTTG TTTTCCAGGA2601 ATTTACTGAT GGCTCCTTTA CTCAGCCCTT ATACCGTGGA GAACTAAATG2651 AACATTTGGG CCTCCTCGGC CCATATATAA GAGCAGAAGT TGAAGATAAT2701 ATCATGGTAA CTTTCAGAAA TCAGGCCTCT CGTCCCTATT CCTTCTATTC2751 TAGCCTTATT TCTTATGAGG AAGATCAGAG GCAAGGAGCA GAACCTAGAA2801 AAAACTTTGT CAAGCCTAAT GAAACCAAAA CTTACTTTTG GAAAGTGCAA2851 CATCATATGG CACCCACTAA AGATGAGTTT GACTGCAAAG CCTGGGCTTA2901 TTTCTCTGAT GTTGACCTGG AAAAAGATGT GCACTCAGGC CTGATTGGAC2951 CCCTTCTGGT CTGCCACACT AACACACTGA ACCCTGCTCA TGGGAGACAA3001 GTGACAGTAC AGGAATTTGC TCTGTTTTTC ACCATCTTTG ATGAGACCAA3051 AAGCTGGTAC TTCACTGAAA ATATGGAAAG AAACTGCAGG GCTCCCTGCA3101 ATATCCAGAT GGAAGATCCC ACTTTTAAAG AGAATTATCG CTTCCATGCA3151 ATCAATGGCT ACATAATGGA TACACTACCT GGCTTAGTAA TGGCTCAGGA3201 TCAAAGGATT CGATGGTATC TGCTCAGCAT GGGCAGCAAT GAAAACATCC3251 ATTCTATTCA TTTCAGTGGA CATGTGTTCA CTGTACGAAA AAAAGAGGAG3301 TATAAAATGG CACTGTACAA TCTCTATCCA GGTGTTTTTG AGACAGTGGA3351 AATGTTACCA TCCAAAGCTG GAATTTGGCG GGTGGAATGC CTTATTGGCG3401 AGCATCTACA TGCTGGGATG AGCACACTTT TTCTGGTGTA CAGCAATAAG3451 TGTCAGACTC CCCTGGGAAT GGCTTCTGGA CACATTAGAG ATTTTCAGAT3501 TACAGCTTCA GGACAATATG GACAGTGGGC CCCAAAGCTG GCCAGACTTC3551 ATTATTCCGG ATCAATCAAT GCCTGGAGCA CCAAGGAGCC CTTTTCTTGG3601 ATCAAGGTGG ATCTGTTGGC ACCAATGATT ATTCACGGCA TCAAGACCCA3651 GGGTGCCCGT CAGAAGTTCT CCAGCCTCTA CATCTCTCAG TTTATCATCA3701 TGTATAGTCT TGATGGGAAG AAGTGGCAGA CTTATCGAGG AAATTCCACT3751 GGAACCTTAA TGGTCTTCTT TGGCAATGTG GATTCATCTG GGATAAAACA3801 CAATATTTTT AACCCTCCAA TTATTGCTCG ATACATCCGT TTGCACCCAA3851 CTCATTATAG CATTCGCAGC ACTCTTCGCA TGGAGTTGAT GGGCTGTGAT3901 TTAAATAGTT GCAGCATGCC ATTGGGAATG GAGAGTAAAG CAATATCAGA3951 TGCACAGATT ACTGCTTCAT CCTACTTTAC CAATATGTTT GCCACCTGGT4001 CTCCTTCAAA AGCTCGACTT CACCTCCAAG GGAGGAGTAA TGCCTGGAGA4051 CCTCAGGTGA ATAATCCAAA AGAGTGGCTG CAAGTGGACT TCCAGAAGAC4101 AATGAAAGTC ACAGGAGTAA CTACTCAGGG AGTAAAATCT CTGCTTACCA4151 GCATGTATGT GAAGGAGTTC CTCATCTCCA GCAGTCAAGA TGGCCATCAG4201 TGGACTCTCT TTTTTCAGAA TGGCAAAGTA AAGGTTTTTC AGGGAAATCA4251 AGACTCCTTC ACACCTGTGG TGAACTCTCT AGACCCACCG TTACTGACTC4301 GCTACCTTCG AATTCACCCC CAGAGTTGGG TGCACCAGAT TGCCCTGAGG4351 ATGGAGGTTC TGGGCTGCGA GGCACAGGAC CTCTACTGA

TABLE 18 Protein sequence of FVIII 265 (FVIII singlechain molecule with pentaglycines at N-terminus; pentaglycine shown in bold) (SEQ ID NO: 103)   1 MQIELSTCFF LCLLRFCFSG GGGGATRRYY LGAVELSWDY MQSDLGELPV  51 DARFPPRVPK SFPFNTSVVY KKTLFVEFTD HLFNIAKPRP PWMGLLGPTI 101 QAEVYDTVVI TLKNMASHPV SLHAVGVSYW KASEGAEYDD QTSQREKEDD 151 KVFPGGSHTY VWQVLKENGP MASDPLCLTY SYLSHVDLVK DLNSGLIGAL 201 LVCREGSLAK EKTQTLHKFI LLFAVFDEGK SWHSETKNSL MQDRDAASAR 251 AWPKMHTVNG YVNRSLPGLI GCHRKSVYWH VIGMGTTPEV HSIFLEGHTF 301 LVRNHRQASL EISPITFLTA QTLLMDLGQF LLFCHISSHQ HDGMEAYVKV 351 DSCPEEPQLR MKNNEEAEDY DDDLTDSEMD WRFDDDNSP SFIQIRSVAK 401 KHPKTWVHYI AAEEEDWDYA PLVLAPDDRS YKSQYLNNGP QRIGRKYKKV 451 RFMAYTDETF KTREAIQHES GILGPLLYGE VGDTLLIIFK NQASRPYNIY 501 PHGITDVRPL YSRRLPKGVK HLKDFPILPG EIFKYKWTVT VEDGPTKSDP 551 RCLTRYYSSF VNMERDLASG LIGPLLICYK ESVDQRGNQI MSDKRNVILF 601 SVFDENRSWY LTENIQRFLP NPAGVQLEDP EFQASNIMHS INGYVFDSLQ 651 LSVCLHEVAY WYILSIGAQT DFLSVFFSGY TFKHKMVYED TLTLFPFSGE 701 TVFMSMENPG LWILGCHNSD FRNRGMTALL KVSSCDKNTG DYYEDSYEDI 751 SAYLLSKNNA IEPRSFSQNP PVLKAHQAEI TRTTLQSDQE EIDYDDTISV 801 EMKKEDFDIY DEDENQSPRS FQKKTRHYFI AAVERLWDYG MSSSPHVLRN 851 RAQSGSVPQF KKVVFQEFTD GSFTQPLYRG ELNEHLGLLG PYIRAEVEDN 901 IMVTFRNQAS RPYSFYSSLI SYEEDQRQGA EPRKNFVKPN ETKTYFWKVQ 951 HHMAPTKDEF DCKAWAYFSD VDLEKDVHSG LIGPLLVCHT NTLNPAHGRQ1001 VTVQEFALFF TIFDETKSWY FTENMERNCR APCNIQMEDP TFKENYRFHA1051 INGYIMDTLP GLVMAQDQRI RWYLLSMGSN ENIHSIHFSG HVFTVRKKEE1101 YKMALYNLYP GVFETVEMLP SKAGIWRVEC LIGEHLHAGM STLFLVYSNK1151 CQTPLGMASG HIRDFQITAS GQYGQWAPKL ARLHYSGSIN AWSTKEPFSW1201 IKVDLLAPMI IHGIKTQGAR QKFSSLYISQ FIIMYSLDGK KWQTYRGNST1251 GTLMVFEGNV DSSGIKHNIF NPPIIARYIR LHPTHYSIRS TLRMELMGCD1301 LNSCSMPLGM ESKAISDAQI TASSYFTNMF ATWSPSKARL HLQGRSNAWR1351 PQVNNPKEWL QVDFQKTMKV TGVTTQGVKS LLTSMYVKEF LISSSQDGHQ1401 WTLFFQNGKV KVFQGNQDSF TPVVNSLDPP LLTRYLRIHP QSWVHQIALR1451 MEVLGCEAQD LY*

Example 20: Plasma Stability and PK of FVIII198 in HemA and FVIII/VWFDouble Knockout (DKO) Plasma

The Plasma stability of FVIII 198 (which is a partial B-domaincontaining single chain FVIIIFc molecule-226N6; where 226 represents theN-terminus 226 amino acids of FVIII B-domain and N6 represents sixN-glycosylation sites in the B-domain) was compared to single chainFVIIIFc (FVIII 155/Fc) in FVIII/VWF double knockout (DKO) plasma.Schematic representation of FVIII155 and FVIII198 can be seen in FIG. 25.

For the stability assay, 5 IU/ml of FVIII 198 or FVIIIFc proteins wasincubated with mouse or DKO plasma at 37° C. Aliquots were collected atdifferent time points for activity measurement by FVIII chromogenicassay. Activity at each time point was measured in duplicate and averageactivity was plotted as a function of time. In the stability assay, thepresence of partial B-domain increased the stability of single chainFVIIIFc (FIG. 26A).

The half-life of FVIII 198 (single chain-B226N6) was also compared withFVIII155 (single chain B-domain deleted FVIII) in DKO mice. FVIII 198has at least about a 1.5 fold longer half-life compared to FVIII155(FIG. 26B). These experiments suggest that there might be a co-relationbetween FVIII stability and its in-vivo half-life.

FVIII198 nucleotide sequence (FVIIIFc with partial B-domain. 226N6)(SEQ ID NO: 104) 1ATGCAAATAG AGCTCTCCAC CTGCTTCTTT CTGTGCCTTT TGCGATTCTG 51CTTTAGTGCC ACCAGAAGAT ACTACCTGGG TGCAGTGGAA CTGTCATGGG 101ACTATATGCA AAGTGATCTC GGTGAGCTGC CTGTGGACGC AAGATTTCCT 151CCTAGAGTGC CAAAATCTTT TCCATTCAAC ACCTCAGTCG TGTACAAAAA 201GACTCTGTTT GTAGAATTCA CGGATCACCT TTTCAACATC GCTAAGCCAA 251GGCCACCCTG GATGGGTCTG CTAGGTCCTA CCATCCAGGC TGAGGTTTAT 301GATACAGTGG TCATTACACT TAAGAACATG GCTTCCCATC CTGTCAGTCT 351TCATGCTGTT GGTGTATCCT ACTGGAAAGC TTCTGAGGGA GCTGAATATG 401ATGATCAGAC CAGTCAAAGG GAGAAAGAAG ATGATAAAGT CTTCCCTGGT 451GGAAGCCATA CATATGTCTG GCAGGTCCTG AAAGAGAATG GTCCAATGGC 501CTCTGACCCA CTGTGCCTTA CCTACTCATA TCTTTCTCAT GTGGACCTGG 551TAAAAGACTT GAATTCAGGC CTCATTGGAG CCCTACTAGT ATGTAGAGAA 601GGGAGTCTGG CCAAGGAAAA GACACAGACC TTGCACAAAT TTATACTACT 651TTTTGCTGTA TTTGATGAAG GGAAAAGTTG GCACTCAGAA ACAAAGAACT 701CCTTGATGCA GGATAGGGAT GCTGCATCTG CTCGGGCCTG GCCTAAAATG 751CACACAGTCA ATGGTTATGT AAACAGGTCT CTGCCAGGTC TGATTGGATG 801CCACAGGAAA TCAGTCTATT GGCATGTGAT TGGAATGGGC ACCACTCCTG 851AAGTGCACTC AATATTCCTC GAAGGTCACA CATTTCTTGT GAGGAACCAT 901CGCCAGGCGT CCTTGGAAAT CTCGCCAATA ACTTTCCTTA CTGCTCAAAC 951ACTCTTGATG GACCTTGGAC AGTTTCTACT GTTTTGTCAT ATCTCTTCCC 1001ACCAACATGA TGGCATGGAA GCTTATGTCA AAGTAGACAG CTGTCCAGAG 1051GAACCCCAAC TACGAATGAA AAATAATGAA GAAGCGGAAG ACTATGATGA 1101TGATCTTACT GATTCTGAAA TGGATGTGGT CAGGTTTGAT GATGACAACT 1151CTCCTTCCTT TATCCAAATT CGCTCAGTTG CCAAGAAGCA TCCTAAAACT 1201TGGGTACATT ACATTGCTGC TGAAGAGGAG GACTGGGACT ATGCTCCCTT 1251AGTCCTCGCC CCCGATGACA GAAGTTATAA AAGTCAATAT TTGAACAATG 1301GCCCTCAGCG GATTGGTAGG AAGTACAAAA AAGTCCGATT TATGGCATAC 1351ACAGATGAAA CCTTTAAGAC TCGTGAAGCT ATTCAGCATG AATCAGGAAT 1401CTTGGGACCT TTACTTTATG GGGAAGTTGG AGACACACTG TTGATTATAT 1451TTAAGAATCA AGCAAGCAGA CCATATAACA TCTACCCTCA CGGAATCACT 1501GATGTCCGTC CTTTGTATTC AAGGAGATTA CCAAAAGGTG TAAAACATTT 1551GAAGGATTTT CCAATTCTGC CAGGAGAAAT ATTCAAATAT AAATGGACAG 1601TGACTGTAGA AGATGGGCCA ACTAAATCAG ATCCTCGGTG CCTGACCCGC 1651TATTACTCTA GTTTCGTTAA TATGGAGAGA GATCTAGCTT CAGGACTCAT 1701TGGCCCTCTC CTCATCTGCT ACAAAGAATC TGTAGATCAA AGAGGAAACC 1751AGATAATGTC AGACAAGAGG AATGTCATCC TGTTTTCTGT ATTTGATGAG 1801AACCGAAGCT GGTACCTCAC AGAGAATATA CAACGCTTTC TCCCCAATCC 1851AGCTGGAGTG CAGCTTGAGG ATCCAGAGTT CCAAGCCTCC AACATCATGC 1901ACAGCATCAA TGGCTATGTT TTTGATAGTT TGCAGTTGTC AGTTTGTTTG 1951CATGAGGTGG CATACTGGTA CATTCTAAGC ATTGGAGCAC AGACTGACTT 2001CCTTTCTGTC TTCTTCTCTG GATATACCTT CAAACACAAA ATGGTCTATG 2051AAGACACACT CACCCTATTC CCATTCTCAG GAGAAACTGT CTTCATGTCG 2101ATGGAAAACC CAGGTCTATG GATTCTGGGG TGCCACAACT CAGACTTTCG 2151GAACAGAGGC ATGACCGCCT TACTGAAGGT TTCTAGTTGT GACAAGAACA 2201CTGGTGATTA TTACGAGGAC AGTTATGAAG ATATTTCAGC ATACTTGCTG 2251AGTAAAAACA ATGCCATTGA ACCAAGAAGC TTCTCTCAGA ATTCAAGACA 2301CCCTAGCACT AGGCAAAAGC AATTTAATGC CACCACAATT CCAGAAAATG 2351ACATAGAGAA GACTGACCCT TGGTTTGCAC ACAGAACACC TATGCCTAAA 2401ATACAAAATG TCTCCTCTAG TGATTTGTTG ATGCTCTTGC GACAGAGTCC 2451TACTCCACAT GGGCTATCCT TATCTGATCT CCAAGAAGCC AAATATGAGA 2501CTTTTTCTGA TGATCCATCA CCTGGAGCAA TAGACAGTAA TAACAGCCTG 2551TCTGAAATGA CACACTTCAG GCCACAGCTC CATCACAGTG GGGACATGGT 2601ATTTACCCCT GAGTCAGGCC TCCAATTAAG ATTAAATGAG AAACTGGGGA 2651CAACTGCAGC AACAGAGTTG AAGAAACTTG ATTTCAAAGT TTCTAGTACA 2701TCAAATAATC TGATTTCAAC AATTCCATCA GACAATTTGG CAGCAGGTAC 2751TGATAATACA AGTTCCTTAG GACCCCCAAG TATGCCAGTT CATTATGATA 2801GTCAATTAGA TACCACTCTA TTTGGCAAAA AGTCATCTCC CCTTACTGAG 2851TCTGGTGGAC CTCTGAGCTT GAGTGAAGAA AATAATGATT CAAAGTTGTT 2901AGAATCAGGT TTAATGAATA GCCAAGAAAG TTCATGGGGA AAAAATGTAT 2951CGTCAGAAAT AACTCGTACT ACTCTTCAGT CAGATCAAGA GGAAATTGAC 3001TATGATGATA CCATATCAGT TGAAATGAAG AAGGAAGATT TTGACATTTA 3051TGATGAGGAT GAAAATCAGA GCCCCCGCAG CTTTCAAAAG AAAACACGAC 3101ACTATTTTAT TGCTGCAGTG GAGAGGCTCT GGGATTATGG GATGAGTAGC 3151TCCCCACATG TTCTAAGAAA CAGGGCTCAG AGTGGCAGTG TCCCTCAGTT 3201CAAGAAAGTT GTTTTCCAGG AATTTACTGA TGGCTCCTTT ACTCAGCCCT 3251TATACCGTGG AGAACTAAAT GAACATTTGG GACTCCTGGG GCCATATATA 3301AGAGCAGAAG TTGAAGATAA TATCATGGTA ACTTTCAGAA ATCAGGCCTC 3351TCGTCCCTAT TCCTTCTATT CTAGCCTTAT TTCTTATGAG GAAGATCAGA 3401GGCAAGGAGC AGAACCTAGA AAAAACTTTG TCAAGCCTAA TGAAACCAAA 3451ACTTACTTTT GGAAAGTGCA ACATCATATG GCACCCACTA AAGATGAGTT 3501TGACTGCAAA GCCTGGGCTT ATTTCTCTGA TGTTGACCTG GAAAAAGATG 3551TGCACTCAGG CCTGATTGGA CCCCTTCTGG TCTGCCACAC TAACACACTG 3601AACCCTGCTC ATGGGAGACA AGTGACAGTA CAGGAATTTG CTCTGTTTTT 3651CACCATCTTT GATGAGACCA AAAGCTGGTA CTTCACTGAA AATATGGAAA 3701GAAACTGCAG GGCTCCCTGC AATATCCAGA TGGAAGATCC CACTTTTAAA 3751GAGAATTATC GCTTCCATGC AATCAATGGC TACATAATGG ATACACTACC 3801TGGCTTAGTA ATGGCTCAGG ATCAAAGGAT TCGATGGTAT CTGCTCAGCA 3851TGGGCAGCAA TGAAAACATC CATTCTATTC ATTTCAGTGG ACATGTGTTC 3901ACTGTACGAA AAAAAGAGGA GTATAAAATG GCACTGTACA ATCTCTATCC 3951AGGTGTTTTT GAGACAGTGG AAATGTTACC ATCCAAAGCT GGAATTTGGC 4001GGGTGGAATG CCTTATTGGC GAGCATCTAC ATGCTGGGAT GAGCACACTT 4051TTTCTGGTGT ACAGCAATAA GTGTCAGACT CCCCTGGGAA TGGCTTCTGG 4101ACACATTAGA GATTTTCAGA TTACAGCTTC AGGACAATAT GGACAGTGGG 4151CCCCAAAGCT GGCCAGACTT CATTATTCCG GATCAATCAA TGCCTGGAGC 4201ACCAAGGAGC CCTTTTCTTG GATCAAGGTG GATCTGTTGG CACCAATGAT 4251TATTCACGGC ATCAAGACCC AGGGTGCCCG TCAGAAGTTC TCCAGCCTCT 4301ACATCTCTCA GTTTATCATC ATGTATAGTC TTGATGGGAA GAAGTGGCAG 4351ACTTATCGAG GAAATTCCAC TGGAACCTTA ATGGTCTTCT TTGGCAATGT 4401GGATTCATCT GGGATAAAAC ACAATATTTT TAACCCTCCA ATTATTGCTC 4451GATACATCCG TTTGCACCCA ACTCATTATA GCATTCGCAG CACTCTTCGC 4501ATGGAGTTGA TGGGCTGTGA TTTAAATAGT TGCAGCATGC CATTGGGAAT 4551GGAGAGTAAA GCAATATCAG ATGCACAGAT TACTGCTTCA TCCTACTTTA 4601CCAATATGTT TGCCACCTGG TCTCCTTCAA AAGCTCGACT TCACCTCCAA 4651GGGAGGAGTA ATGCCTGGAG ACCTCAGGTG AATAATCCAA AAGAGTGGCT 4701GCAAGTGGAC TTCCAGAAGA CAATGAAAGT CACAGGAGTA ACTACTCAGG 4751GAGTAAAATC TCTGCTTACC AGCATGTATG TGAAGGAGTT CCTCATCTCC 4801AGCAGTCAAG ATGGCCATCA GTGGACTCTC TTTTTTCAGA ATGGCAAAGT 4851AAAGGTTTTT CAGGGAAATC AAGACTCCTT CACACCTGTG GTGAACTCTC 4901TAGACCCACC GTTACTGACT CGCTACCTTC GAATTCACCC CCAGAGTTGG 4951GTGCACCAGA TTGCCCTGAG GATGGAGGTT CTGGGCTGCG AGGCACAGGA 5001CCTCTACGAC AAAACTCACA CATGCCCACC GTGCCCAGCT CCAGAACTCC 5051TGGGCGGACC GTCAGTCTTC CTCTTCCCCC CAAAACCCAA GGACACCCTC 5101ATGATCTCCC GGACCCCTGA GGTCACATGC GTGGTGGTGG ACGTGAGCCA 5151CGAAGACCCT GAGGTCAAGT TCAACTGGTA CGTGGACGGC GTGGAGGTGC 5201ATAATGCCAA GACAAAGCCG CGGGAGGAGC AGTACAACAG CACGTACCGT 5251GTGGTCAGCG TCCTCACCGT CCTGCACCAG GACTGGCTGA ATGGCAAGGA 5301GTACAAGTGC AAGGTCTCCA ACAAAGCCCT CCCAGCCCCC ATCGAGAAAA 5351CCATCTCCAA AGCCAAAGGG CAGCCCCGAG AACCACAGGT GTACACCCTG 5401CCCCCATCCC GGGATGAGCT GACCAAGAAC CAGGTCAGCC TGACCTGCCT 5451GGTCAAAGGC TTCTATCCCA GCGACATCGC CGTGGAGTGG GAGAGCAATG 5501GGCAGCCGGA GAACAACTAC AAGACCACGC CTCCCGTGTT GGACTCCGAC 5551GGCTCCTTCT TCCTCTACAG CAAGCTCACC GTGGACAAGA GCAGGTGGCA 5601GCAGGGGAAC GTCTTCTCAT GCTCCGTGAT GCATGAGGCT CTGCACAACC 5651ACTACACGCA GAAGAGCCTC TCCCTGTCTC CGGGTAAATG A FVIII 198 protein sequence(SEQ ID NO: 105) 1MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQSDL GELPVDARFP 51PRVPKSFPFN TSVVYKKTLF VEFTDHLFNI AKPRPPWMGL LGPTIQAEVY 101DTVVITLKNM ASHPVSLHAV GVSYWKASEG AEYDDQTSQR EKEDDKVFPG 151GSHTYVWQVL KENGPMASDP LCLTYSYLSH VDLVKDLNSG LIGALLVCRE 201GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD AASARAWPKM 251HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL EGHTFLVRNH 301RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME AYVKVDSCPE 351EPQLRMKNNE EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI RSVAKKHPKT 401WVHYIAAEEE DWDYAPLVLA PDDRSYKSQY LNNGPQRIGR KYKKVRFMAY 451TDETFKTREA IQHESGILGP LLYGEVGDTL LIIFKNQASR PYNIYPHGIT 501DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP TKSDPRCLTR 551YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR NVILFSVFDE 601NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHSINGYV FDSLQLSVCL 651HEVAYWYILS IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF PFSGETVFMS 701MENPGLWILG CHNSDFRNRG MTALLKVSSC DKNTGDYYED SYEDISAYLL 751SKNNAIEPRS FSQNSRHPST RQKQFNATTI PENDIEKTDP WFAHRTPMPK 801IQNVSSSDLL MLLRQSPTPH GLSLSDLQEA KYETFSDDPS PGAIDSNNSL 851SEMTHFRPQL HHSGDMVFTP ESGLQLRLNE KLGTTAATEL KKLDFKVSST 901SNNLISTIPS DNLAAGTDNT SSLGPPSMPV HYDSQLDTTL FGKKSSPLTE 951SGGPLSLSEE NNDSKLLESG LMNSQESSWG KNVSSEITRT TLQSDQEEID 1001YDDTISVEMK KEDFDIYDED ENQSPRSFQK KTRHYFIAAV ERLWDYGMSS 1051SPHVLRNRAQ SGSVPQFKKV VFQEFTDGSF TQPLYRGELN EHLGLLGPYI 1101RAEVEDNIMV TFRNQASRPY SFYSSLISYE EDQRQGAEPR KNFVKPNETK 1151TYFWKVQHHM APTKDEFDCK AWAYFSDVDL EKDVHSGLIG PLLVCHTNTL 1201NPAHGRQVTV QEFALFFTIF DETKSWYFTE NMERNCRAPC NIQMEDPTFK 1251ENYRFHAING YIMDTLPGLV MAQDQRIRWY LLSMGSNENI HSIHFSGHVF 1301TVRKKEEYKM ALYNLYPGVF ETVEMLPSKA GIWRVECLIG EHLHAGMSTL 1351FLVYSNKCQT PLGMASGHIR DFQITASGQY GQWAPKLARL HYSGSINAWS 1401TKEPFSWIKV DLLAPMIIHG IKTQGARQKF SSLYISQFII MYSLDGKKWQ 1451TYRGNSTGTL MVFFGNVDSS GIKHNIFNPP IIARYIRLHP THYSIRSTLR 1501MELMGCDLNS CSMPLGMESK AISDAQITAS SYFTNMFATW SPSKARLHLQ 1551GRSNAWRPQV NNPKEWLQVD FQKTMKVTGV TTQGVKSLLT SMYVKEFLIS 1601SSQDGHQWTL FFQNGKVKVF QGNQDSFTPV VNSLDPPLLT RYLRIHPQSW 1651VHQIALRMEV LGCEAQDLYD KTHTCPPCPA PELLGGPSVF LFPPKPKDTL 1701MISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYNSTYR 1751VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTL 1801PPSRDELTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD 1851GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK*

Example 21. Expression of D1D2 Protein of VWF

Proper folding of D′D3 domain is essential for its binding to FVIII. VWFpropeptide (D1D2-amino acids 1-763) is required for efficient disulfidebond formation and folding of D′D3. It acts as an internal chaperone forD′D3 folding. VWF constructs making VWF fragments can either beexpressed where VWF propeptide (i.e. D1D2 domain) is directly attachedto D′D3 domain and removed during the regular intracellular processingof D′D3 (i.e. in cis) or, it can either be expressed from other plasmidi.e. in trans. We designed FVIII-VWF heterodimer in such a way whereD1D2 can either be expressed in cis or trans.

Cloning VWF 053: VWF 053 clone expresses VWF propeptide (D1D2 domain)for in trans expression of D1D2. VWF propeptide was PCR amplified fromfull length using ESC 54 and ESC124.

ESC54-VWF forward with BsiW1 site (SEQ ID NO: 111)(CGCTTCGCGACGTACGGCCGCCACCATGATT CCTGCCAGATTTGCCGGGGTGCTGCTTGCTC)ESC 124-D1D2 cloning oligo with Not1 site-reverse (SEQ ID NO: 112)(CTAGACTCGAGCGGCCGCTCACCTTTTGCTG CGATGAGACAGGGGACTGCTGAGGACAGC)

PCR product was digested with BsiW1 and Not1 and ligated into BsiW1/Not1digested pCDNA 4.

Nucleotide sequence of VWF 053 (VWF D1D2-propeptide) (SEQ ID NO: 113) 1ATGATTCCTG CCAGATTTGC CGGGGTGCTG CTTGCTCTGG CCCTCATTTT 51GCCAGGGACC CTTTGTGCAG AAGGAACTCG CGGCAGGTCA TCCACGGCCC 101GATGCAGCCT TTTCGGAAGT GACTTCGTCA ACACCTTTGA TGGGAGCATG 151TACAGCTTTG CGGGATACTG CAGTTACCTC CTGGCAGGGG GCTGCCAGAA 201ACGCTCCTTC TCGATTATTG GGGACTTCCA GAATGGCAAG AGAGTGAGCC 251TCTCCGTGTA TCTTGGGGAA TTTTTTGACA TCCATTTGTT TGTCAATGGT 301ACCGTGACAC AGGGGGACCA AAGAGTCTCC ATGCCCTATG CCTCCAAAGG 351GCTGTATCTA GAAACTGAGG CTGGGTACTA CAAGCTGTCC GGTGAGGCCT 401ATGGCTTTGT GGCCAGGATC GATGGCAGCG GCAACTTTCA AGTCCTGCTG 451TCAGACAGAT ACTTCAACAA GACCTGCGGG CTGTGTGGCA ACTTTAACAT 501CTTTGCTGAA GATGACTTTA TGACCCAAGA AGGGACCTTG ACCTCGGACC 551CTTATGACTT TGCCAACTCA TGGGCTCTGA GCAGTGGAGA ACAGTGGTGT 601GAACGGGCAT CTCCTCCCAG CAGCTCATGC AACATCTCCT CTGGGGAAAT 651GCAGAAGGGC CTGTGGGAGC AGTGCCAGCT TCTGAAGAGC ACCTCGGTGT 701TTGCCCGCTG CCACCCTCTG GTGGACCCCG AGCCTTTTGT GGCCCTGTGT 751GAGAAGACTT TGTGTGAGTG TGCTGGGGGG CTGGAGTGCG CCTGCCCTGC 801CCTCCTGGAG TACGCCCGGA CCTGTGCCCA GGAGGGAATG GTGCTGTACG 851GCTGGACCGA CCACAGCGCG TGCAGCCCAG TGTGCCCTGC TGGTATGGAG 901TATAGGCAGT GTGTGTCCCC TTGCGCCAGG ACCTGCCAGA GCCTGCACAT 951CAATGAAATG TGTCAGGAGC GATGCGTGGA TGGCTGCAGC TGCCCTGAGG 1001GACAGCTCCT GGATGAAGGC CTCTGCGTGG AGAGCACCGA GTGTCCCTGC 1051GTGCATTCCG GAAAGCGCTA CCCTCCCGGC ACCTCCCTCT CTCGAGACTG 1101CAACACCTGC ATTTGCCGAA ACAGCCAGTG GATCTGCAGC AATGAAGAAT 1151GTCCAGGGGA GTGCCTTGTC ACTGGTCAAT CCCACTTCAA GAGCTTTGAC 1201AACAGATACT TCACCTTCAG TGGGATCTGC CAGTACCTGC TGGCCCGGGA 1251TTGCCAGGAC CACTCCTTCT CCATTGTCAT TGAGACTGTC CAGTGTGCTG 1301ATGACCGCGA CGCTGTGTGC ACCCGCTCCG TCACCGTCCG GCTGCCTGGC 1351CTGCACAACA GCCTTGTGAA ACTGAAGCAT GGGGCAGGAG TTGCCATGGA 1401TGGCCAGGAC ATCCAGCTCC CCCTCCTGAA AGGTGACCTC CGCATCCAGC 1451ATACAGTGAC GGCCTCCGTG CGCCTCAGCT ACGGGGAGGA CCTGCAGATG 1501GACTGGGATG GCCGCGGGAG GCTGCTGGTG AAGCTGTCCC CCGTCTATGC 1551CGGGAAGACC TGCGGCCTGT GTGGGAATTA CAATGGCAAC CAGGGCGACG 1601ACTTCCTTAC CCCCTCTGGG CTGGCGGAGC CCCGGGTGGA GGACTTCGGG 1651AACGCCTGGA AGCTGCACGG GGACTGCCAG GACCTGCAGA AGCAGCACAG 1701CGATCCCTGC GCCCTCAACC CGCGCATGAC CAGGTTCTCC GAGGAGGCGT 1751GCGCGGTCCT GACGTCCCCC ACATTCGAGG CCTGCCATCG TGCCGTCAGC 1801CCGCTGCCCT ACCTGCGGAA CTGCCGCTAC GACGTGTGCT CCTGCTCGGA 1851CGGCCGCGAG TGCCTGTGCG GCGCCCTGGC CAGCTATGCC GCGGCCTGCG 1901CGGGGAGAGG CGTGCGCGTC GCGTGGCGCG AGCCAGGCCG CTGTGAGCTG 1951AACTGCCCGA AAGGCCAGGT GTACCTGCAG TGCGGGACCC CCTGCAACCT 2001GACCTGCCGC TCTCTCTCTT ACCCGGATGA GGAATGCAAT GAGGCCTGCC 2051TGGAGGGCTG CTTCTGCCCC CCAGGGCTCT ACATGGATGA GAGGGGGGAC 2101TGCGTGCCCA AGGCCCAGTG CCCCTGTTAC TATGACGGTG AGATCTTCCA 2151GCCAGAAGAC ATCTTCTCAG ACCATCACAC CATGTGCTAC TGTGAGGATG 2201GCTTCATGCA CTGTACCATG AGTGGAGTCC CCGGAAGCTT GCTGCCTGAC 2251GCTGTCCTCA GCAGTCCCCT GTCTCATCGC AGCAAAAGGProtein sequence of VWF 053 (VWF D1D2-Propeptide) (SEQ ID NO: 114) 1MIPARFAGVL LALALILPGT LCAEGTRGRS STARCSLFGS DFVNTFDGSM 51YSFAGYCSYL LAGGCQKRSF SIIGDFQNGK RVSLSVYLGE FFDIHLFVNG 101TVTQGDQRVS MPYASKGLYL ETEAGYYKLS GEAYGFVARI DGSGNFQVLL 151SDRYFNKTCG LCGNFNIFAE DDFMTQEGTL TSDPYDFANS WALSSGEQWC 201ERASPPSSSC NISSGEMQKG LWEQCQLLKS TSVFARCHPL VDPEPFVALC 251EKTLCECAGG LECACPALLE YARTCAQEGM VLYGWTDHSA CSPVCPAGME 301YRQCVSPCAR TCQSLHINEM CQERCVDGCS CPEGQLLDEG LCVESTECPC 351VHSGKRYPPG TSLSRDCNTC ICRNSQWICS NEECPGECLV TGQSHFKSFD 401NRYFTFSGIC QYLLARDCQD HSFSIVIETV QCADDRDAVC TRSVTVRLPG 451LHNSLVKLKH GAGVAMDGQD IQLPLLKGDL RIQHTVTASV RLSYGEDLQM 501DWDGRGRLLV KLSPVYAGKT CGLCGNYNGN QGDDFLTPSG LAEPRVEDFG 551NAWKLHGDCQ DLQKQHSDPC ALNPRMTRFS EEACAVLTSP TFEACHRAVS 601PLPYLRNCRY DVCSCSDGRE CLCGALASYA AACAGRGVRV AWREPGRCEL 651NCPKGQVYLQ CGTPCNLTCR SLSYPDEECN EACLEGCFCP PGLYMDERGD 701CVPKAQCPCY YDGEIFQPED IFSDHHTMCY CEDGFMHCTM SGVPGSLLPD 751AVLSSPLSHR SKR

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingknowledge within the skill of the art, readily modify and/or adapt forvarious applications such specific embodiments, without undueexperimentation, without departing from the general concept of thepresent invention. Therefore, such adaptations and modifications areintended to be within the meaning and range of equivalents of thedisclosed embodiments, based on the teaching and guidance presentedherein. It is to be understood that the phraseology or terminologyherein is for the purpose of description and not of limitation, suchthat the terminology or phraseology of the present specification is tobe interpreted by the skilled artisan in light of the teachings andguidance.

Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

All patents and publications cited herein are incorporated by referenceherein in their entirety.

1-147. (canceled)
 148. A chimeric protein comprising a first polypeptideand a second polypeptide, wherein the first polypeptide comprises: (a) aFactor VIII (“FVIII”) protein comprising amino acid residues 1 to 740 ofSEQ ID NO: 16 and a B-domain deletion; and (b) a first immunoglobulinconstant region, wherein the second polypeptide comprises: (a) a vonWillebrand Factor (VWF) fragment comprising a D′ and a D3 domain of VWF,wherein the VWF fragment comprises an alanine substitution for cysteineat residues corresponding to residue 1099 and residue 1142 of SEQ ID NO:2; (b) a second immunoglobulin constant region; and (c) a cleavablelinker located between the VWF fragment and the second immunoglobulinconstant region, wherein the first polypeptide and the secondpolypeptide are linked by a disulfide bond between the first and secondimmunoglobulin constant regions or the portions thereof.
 149. Apolynucleotide encoding a chimeric protein comprising a firstpolypeptide and a second polypeptide, wherein the first polypeptidecomprises: (a) a Factor VIII (“FVIII”) protein comprising amino acidresidues 1 to 740 of SEQ ID NO: 16 and a B-domain deletion; and (b) afirst immunoglobulin constant region, wherein the second polypeptidecomprises: (a) a von Willebrand Factor (VWF) fragment comprising a D′and a D3 domain of VWF, wherein the VWF fragment comprises an alaninesubstitution for cysteine at residues corresponding to residue 1099 andresidue 1142 of SEQ ID NO: 2; (b) a second immunoglobulin constantregion; and (c) a cleavable linker located between the VWF fragment andthe second immunoglobulin constant region, wherein the first polypeptideand the second polypeptide are linked by a disulfide bond between thefirst and second immunoglobulin constant regions or the portionsthereof.
 150. A host cell comprising a polynucleotide encoding achimeric protein comprising a first polypeptide and a secondpolypeptide, wherein the first polypeptide comprises: (a) a Factor VIII(“FVIII”) protein comprising amino acid residues 1 to 740 of SEQ ID NO:16 and a B-domain deletion; and (b) a first immunoglobulin constantregion, wherein the second polypeptide comprises: (a) a von WillebrandFactor (VWF) fragment comprising a D′ and a D3 domain of VWF, whereinthe VWF fragment comprises an alanine substitution for cysteine atresidues corresponding to residue 1099 and residue 1142 of SEQ ID NO: 2;(b) a second immunoglobulin constant region; and (c) a cleavable linkerlocated between the VWF fragment and the second immunoglobulin constantregion, wherein the first polypeptide and the second polypeptide arelinked by a disulfide bond between the first and second immunoglobulinconstant regions or the portions thereof.
 151. A method of treatinghemophilia A in a subject in need thereof comprising administering aneffective amount of a chimeric protein comprising a first polypeptideand a second polypeptide, wherein the first polypeptide comprises: (a) aFactor VIII (“FVIII”) protein comprising amino acid residues 1 to 740 ofSEQ ID NO: 16 and a B-domain deletion; and (b) a first immunoglobulinconstant region, wherein the second polypeptide comprises: (a) a vonWillebrand Factor (VWF) fragment comprising a D′ and a D3 domain of VWF,wherein the VWF fragment comprises an alanine substitution for cysteineat residues corresponding to residue 1099 and residue 1142 of SEQ ID NO:2; (b) a second immunoglobulin constant region; and (c) a cleavablelinker located between the VWF fragment and the second immunoglobulinconstant region, wherein the first polypeptide and the secondpolypeptide are linked by a disulfide bond between the first and secondimmunoglobulin constant regions or the portions thereof.
 152. The methodof claim 151, wherein the chimeric protein is administeredintravenously.
 153. A method of treating von Willebrand's disease (VWD)in a subject in need thereof comprising administering an effectiveamount of a chimeric protein comprising a first polypeptide and a secondpolypeptide, wherein the first polypeptide comprises: (a) a Factor VIII(“FVIII”) protein comprising amino acid residues 1 to 740 of SEQ ID NO:16 and a B-domain deletion; and (b) a first immunoglobulin constantregion, wherein the second polypeptide comprises: (a) a von WillebrandFactor (VWF) fragment comprising a D′ and a D3 domain of VWF, whereinthe VWF fragment comprises an alanine substitution for cysteine atresidues corresponding to residue 1099 and residue 1142 of SEQ ID NO: 2;(b) a second immunoglobulin constant region; and (c) a cleavable linkerlocated between the VWF fragment and the second immunoglobulin constantregion, wherein the first polypeptide and the second polypeptide arelinked by a disulfide bond between the first and second immunoglobulinconstant regions or the portions thereof.
 154. The method of claim 153,wherein the chimeric protein is administered intravenously.
 155. Themethod of claim 153, wherein the VWD is Type 2N VWD.